Antibodies to cell-cycle regulatory protein p16, and uses related thereto

ABSTRACT

The present invention relates to the discovery in eukaryotic cells, particularly mammalian cells, of a family of cell-cycle regulatory proteins (“CCR-proteins”). As described herein, this family of proteins includes a polypeptide having an apparent molecular weight of 16 kDa, and a polypeptide having an apparent molecular weight of approximately 15 kDa, each of which can function as an inhibitor of cell-cycle progression, and therefore ultimately of cell growth. The present invention comprises antibodies directed to such CCR-proteins. The present invention is directed to a kit for detecting the level of cyclin-dependent kinase inhibitor p16 gene expression comprising antibodies directed to a p16 protein.

This application is a continuation of application Ser. No. 09/016,869,filed on Jan. 30, 1998, which is a continuation of Ser. No. 08/893,274,filed on Jul. 15, 1997, now U.S. Pat. No. 5,968,821; which is acontinuation of Ser. No. 08/306,511, filed Sep. 14, 1994, now U.S. Pat.No. 5,962,316; which is a continuation-in-part of 08/248,812 filed May25, 1994, now U.S. Pat. No. 5,889,169; which is a continuation-in-partof 08/227,371, filed Apr. 14, 1994; which is a continuation-in-part of08/154,915, filed Nov. 18, 1993, now abandoned. All of theabove-identified applications are incorporated herein by reference intheir entirety.

FUNDING

Work described herein was supported by National Institutes of HealthGrant and the Howard Hughes Medical Institute under NIH Grant Nos. R01GM 39620 and R01 CA 63518. The United States Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Neoplasia is characterized by deregulated cell growth and division.Inevitably, molecular pathways controlling cell growth must interactwith those regulating cell division. It was not until very recently,however, that experimental evidence became available to bring suchconnection to light. Cyclin A was found in association with theadenovirus oncoprotein E1A in virally transformed cells (Giordona et al.Cell 58:981 (1989); and Pines et al. Nature 346:760 (1990)). In an earlyhepatocellular carcinoma, the human cyclin A gene was found to be theintegration site of a fragment of the hepatitis B virus, which leads toactivation of cyclin A transcription and a chimeric viral cyclin Aprotein that is not degradable in vitro (Wang et al. Nature 343:555(1990)). The cell-cycle gene implicated most strongly in oncogenesisthus far is the human cyclin D1. It was originally isolated throughgenetic complementation of yeast G₁ cyclin deficient strains Niong etal. Cell 65:691 (1991); and Lew et al. Cell 66:1197 (1991)), as cellulargenes whose transcription is stimulated by CSF-1 in murine macrophages(Matsushine et al. Cell 65:701 (1991)) and in the putative oncogenePRADI rearranged in parathyroid tumors (Montokura et al. Nature 350:512(1991). Two additional human D-type cyclins, cyclins D2 and D3, weresubsequently identified using PCR and low-stringency hybridizationtechniques (Inaba et al. Genomics 13:565 (1992); and Xiong et al.Genomics 13:575 (1992)). Cyclin D1 is genetically linked to the bcl-1oncogene, a locus activated by translocation to an immunoglobulin geneenhancer in some B-cell lymphomas and leukemias, and located at a siteof gene amplification in 15-20% of human breast cancers and 25-48% ofsquamous cell cancers of head and neck origin.

However, the creation of a mutant oncogene is only one of therequirements needed for tumor formation; tumorigenesis appears to alsorequire the additional inactivation of a second class of critical genes:the “anti-oncogenes” or “tumor-suppressing genes.” In their naturalstate these genes act to suppress cell proliferation. Damage to suchgenes leads to a loss of this suppression, and thereby results intumorigenesis. Thus, the deregulation of cell growth may be mediated byeither the activation of oncogenes or the inactivation oftumor-suppressing genes (Weinberg, R. A., (September 1988) ScientificAmer. pp 44-51).

Oncogenes and tumor-suppressing genes have a basic distinguishedfeature. The oncogenes identified thus far have arisen only in somaticcells, and thus have been incapable of transmitting their effects to thegerm line of the host animal. In contrast, mutations intumor-suppressing genes can be identified in germ line cells, and arethus transmissible to an animal's progeny.

The classic example of a hereditary cancer is retinoblastomas inchildren. The incidence of the retinoblastomas is determined by a tumorsuppressor gene, the retinoblastoma (RB) gene (Weinberg, R. A.,(September 1988) Scientific Amer. pp 44-51; Hansen et al. (1988) TrendsGenet. 4:125-128). Individuals born with a lesion in one of the RBalleles are predisposed to early childhood development ofretinoblastomas. Inactivation or mutation of the second RB allele in oneof the somatic cells of these susceptible individuals appears to be themolecular event that leads to tumor formation (Caveneee et al. (1983)Nature 305:799-784; Friend et al. (1987) PNAS 84:9059-9063).

The RB tumor-suppressing gene has been localized onto human chromosome13. The mutation may be readily transmitted through the germ line ofafflicted individuals (Cavenee, et al. (1986) New Engl. J. Med314:1201-1207). Individuals who have mutations in only one of the twonaturally present alleles of this tumor-suppressing gene are predisposedto retinoblastoma. Inactivation of the second of the two alleles is,however, required for tumorigenesis (Knudson (1971) PNAS 68:820-823).

A second tumor-suppressing gene is the p53 gene (Green (1989) Cell56:1-3; Mowat et al (1985 Nature 314:633-636). The protein encoded bythe p53 gene is a nuclear protein that forms a stable complex with boththe SV40 large T antigen and the adenovirus E1B 55 kd protein. The p53gene product may be inactivated by binding to these proteins.

Based on cause and effect analysis of p53 mutants, the functional roleof p53 as a “cell-cycle checkpoint”, particularly with respect tocontrolling progression of a cell from G₁ phase into S phase, hasimplicated p53 as able to directly or indirectly affect cycle cylemachinery. The first firm evidence for a specific biochemical linkbetween p53 and the cell-cycle comes a finding that p53 apparentlyregulates expression of a second protein, p21, which inhibitscyclin-dependent kinases (CDKs) needed to drive cells through thecell-cycle, e.g. from G₁ into S phase. For example, it has beendemonstrated that non-viral transformation, such as resulting at leastin part from a mutation of deletion of the p53 tumor suppressor, canresult in loss of p21 from cyclin/CDK complexes. As described Xiong etal. (1993) Nature 366:701-704, induction of p21 in response to p53represents a plausible mechanism for effecting cell-cycle arrest inresponse to DNA damage, and loss of p53 may deregulate growth by loss ofthe p21 cell-cycle inhibitor.

The role of RB as a tumor-suppressor protein in cell-cycle control isbelieved to be similar to that of p53. However, whereas p53 is generallybelieved to be responsive to such indigenous environmental cues as DNAdamage, the RB protein is apparently involved in coordinating cellgrowth with exogenous stimulus that normally persuade a cell to ceaseproliferating, such as diffusible growth inhibitors. In normal cells, RBis expressed throughout the cell cycle but exists in multiplephosphorylated forms that are specific for certain phases of the cycle.The more highly phosphorylated forms are found during S and G₂/M,whereas the underphosphorylated forms are the primary species seen in G₁and in the growth arrested state. Base on these observations, it hasbeen argued that if RB is to have a regulatory (suppressive) activity inthe cell-cycle, this activity must be regulated at thepost-translational level. Accordingly, underphosphorylated RB would bethe form with growth-suppressive activity, since this form is prevalentin G₁ and growth arrested cells.

To this end, it is noted that various paracrine growth inhibitors, suchas members of the TGF-β family, prevent phosphorylation of RB and arrestcells in late G₁. Current models suggest that during G₁, cyclindependent kinases and particularly cyclin D-associated kinases, CDK4 andCDK6, phosphorylate the product of the retinoblastoma susceptibilitygene, RB, and thus release cells from its growth inhibitory effects.TGF-β treatment causes accumulation of RB in the under-phosphorylatedstate and expression of RB-inactivating viral oncoproteins prevent TGF-βinduced cell cycle arrest. In similar fashion, other relateddifferentiation factors, such as activin, induce accumulation ofunphosphorylated RB that is correlated with arrest in G.sub.1 phase.

Recently, it has been demonstrated that the RB protein is aphosphorylation substrate for both CDK4 and CDK6 (Serano et al. (1993)Nature 366:704-707; Kato et al. (1993) Genes Dev 7:331-342; and Meyersonet al. (1994) Mol Cell Biol 14:2077-2086). However, prior to the presentdiscovery, there was little information concerning the manner by whichCDK phosphorylation of RB was negatively regulated.

SUMMARY OF THE INVENTION

The present invention relates to the discovery in eulcaryotic cells,particularly mammalian cells, of a novel family of cell-cycle regulatoryproteins (“CCR-proteins”). As described herein, this family of proteinsincludes a polypeptide having an apparent molecular weight of 16 kDa,and a polypeptide having an apparent molecular weight of approximately15 kDa, each of which can function as an inhibitor of cell-cycleprogression, and therefore ultimately of cell growth. Thus, similar tothe role of p21 to the p53 checkpoint, the subject CCR-proteins mayfunction coordinately with the cell-cycle regulatory protein,retinoblastoma (RB). Furthermore, the CCR-protein family includes aprotein having an apparent molecular weight of 13.5 kDa (hereinafter“p13.5”). The presumptive role of p13.5, like p16 and p15, is in theregulation of the cell-cycle.

One aspect of the invention features a substantially pure preparation ofa cell cycle regulatory (CCR) protein, or a fragment thereof, thefull-length form of the CCR-protein having an approximate molecularweight in the range of 13 to 16.5 kD, preferably 14.5 kD to 16 kD. Inpreferred embodiments, the full length form of the CCR-protein has anapparent molecular weight of approximately 13.5 kD, 15 kD, 15.5 kD or 16kD. In a preferred embodiment: the polypeptide has an amino acidsequence at least 60% homologous to the amino acid sequence representedin one of SEQ ID NO: 2, 4 or 6; the polypeptide has an amino acidsequence at least 80% homologous to the amino acid sequence representedin one of SEQ ID NO: 2, 4 or 6; the polypeptide has an amino acidsequence at least 90% homologous to the amino acid sequence representedin one of SEQ ID NO: 2, 4 or 6; the polypeptide has an amino acidsequence identical to the amino acid sequence represented in one of SEQID NO: 2, 4 or 6. In a preferred embodiment: the fragment comprises atleast 5 contiguous amino acid residues of SEQ ID NO: 2, 4 or 6; thefragment comprises at least 20 contiguous amino acid residues of SEQ IDNO: 2, 4 or 6; the fragment comprises at least 50 contiguous amino acidresidues of SEQ ID NO: 2, 4 or 6. For instance, the CCR-protein cancomprise an amino acid sequence represented by the general formula:

(SEQ ID NO: 11) Met-Met-Met-Gly-Xaa-Xaa-Xaa-Val-Ala-Xaa-Leu-Leu-Leu-Xaa-Xaa- Gly-Ala-Xaa-Xaa-Asn-Cys-Xaa-Asp-Pro-Xaa-Thr-Xaa-Xaa-Xaa-Arg- Pro-Val-His-Asp-Ala-Ala-Arg-Glu-Gly-Phe-Leu-Asp-Thr-Leu-V- al- Val-Leu-His-Xaa-Xaa-Gly-Ala-Arg-Leu-Asp-Val-Arg-Asp-Al-a- Trp-Gly-Arg-Leu-Pro-Xaa-Asp-Leu-Ala-Xaa-Glu-Xaa- Gly-His- -Xaa-Asp-Xaa-Xaa-Xaa-Tyr-Leu-Arg-Xaa- Ala-Xaa-Gly

For example, the CCR-protein can be represented by the sequence:

(SEQ ID NO: 12) Met-Asp-Pro-Ala-Ala-Gly-Ser-Ser-Met-Glu-Pro-Ser-Ala-Asp-Trp-Leu- Ala-Thr-Ala-Ala-Ala-Arg-Gly-Arg-Val-Glu-Glu-Val-Arg-Ala-L- eu-Leu- Glu-Ala-Val-Ala-Leu-Pro-Asn-Ala-Pro-Asn-Ser-Tyr-Gl- y-Arg-Arg- Pro-Ile-Gln-Val-Met-Met-Met-Gly-Xaa-Xaa-Xaa- Val- -Ala-Xaa-Leu-Leu-Leu-Xaa-Xaa-Gly-Ala-Xaa- Xaa-Asn-Cys-Xaa-- Asp-Pro-Xaa-Thr-Xaa-Xaa-Xaa-Arg-Pro-Val-His-Asp-Ala-Ala-A- rg-Glu-Gly-Phe-Leu- Asp-Thr-Leu-Val-Val-Leu-His-Xaa-Xaa-Gly-Ala-Arg- Leu-Asp-Val-Arg-Asp-Ala-Trp-Gly-Arg-Leu-Pro-Xaa- Asp-Leu-Ala-Xaa-Glu-Xaa-Glu-His-Xaa-Asp-Xaa-Xaa-Xaa-Tyr-Leu-Arg-Xaa-Ala-Xaa-Gly-Gly-Thr-Arg-Gly- Ser-Asn-His- -Ala-Arg-Ile-Asp-Ala-Ala-Glu-Gly- Pro-Ser-Asp-Ile-Pro-Asp;

or alternatively, by the sequence:

(SEQ ID NO: 13) Met-Arg-Glu-Glu-Asn-Lys-Gly-Met-Pro-Ser-Gly-Gly-Gly-Ser-Asp-Glu- Gly-Leu-Ala-Thr-Pro-Ala-Arg-Gly-Leu-Val-Glu-Lys-Val-Arg-H- is-Ser- Trp-Glu-Ala-Gly-Ala-Asp-Pro-Asn-Gly-Val-Asn-Arg-Ph- e-Gly-Arg- Arg-Ala-Ile-Gln-Val-Met-Met-Met-Gly-Xaa-Xaa- Xaa- -Val-Ala-Xaa-Leu-Leu-Leu-Xaa-Xaa-Gly-Ala- Xaa-Xaa-Asn-Cys-- Xaa-Asp-Pro-Xaa-Thr-Xaa-Xaa-Xaa-Arg-Pro-Val-His-Asp-Ala-A- la-Arg-Glu-Gly-Phe- Leu-Asp-Thr-Leu-ValVal-Leu-His-Xaa-Xaa-Gly-Ala- Arg-Leu-Asp-Val-Arg-Asp-Ala-Trp-Gly-Arg-Leu-Pro- Xaa-Asp-Leu-Ala-Xaa-Glu-Xaa-Gly-His-Xaa-Asp-Xaa-Xaa-Xaa-Tyr-Leu-Arg-Xaa-Ala-Xaa-Gly-Asp,

or yet in another embodiment, by the sequence:

(SEQ ID NO: 14) Met-Met-Met-Gly-Xaa-Xaa-Xaa-Val-Ala-Xaa-Leu-Leu-Leu-Xaa-Xaa-Gly- Ala-Xaa-Xaa-Asn-Cys-Xaa-Asp-Pro-Xaa-Thr-Xaa-Xaa-Xaa-Arg-P- ro-Val- His-Asp-Ala-Ala-Arg-Glu-Gly-Phe-Leu-Asp-Thr-Leu-Va- l-Val-Leu- His-Xaa-Xaa-Gly-Ala-Arg-Leu-Asp-Val-Arg-Asp- Ala- -Trp-Gly-Arg-Leu-Pro-Xaa-Asp-Leu-Ala-Xaa- Glu-Xaa-Glu-His-- Xaa-Asp-Xaa-Xaa-Xaa-Tyr-Leu-Arg-Xaa-Ala-Xaa-Gly-Cys-Ser-L- eu-Cys-Ser-Ala-Gly- Trp-Ser-Leu-Cys-Thr-Ala-Gly-Asn-Val-Ala-Gln-Thr- Asp-Gly-His-Ser-Phe-Ser-Ser-Ser-Thr-Pro-Arg-Ala- Leu-Glu-Leu-Arg-Gly-Gln-Ser-Gln-Glu-Gln-Ser.

In preferred embodiments, the CCR-protein specifically binds a CDK, e.g.a G.sub.1 phase CDK, e.g. CDK4 and/or CDK6. The CCR-protein can becloned from a mammalian cell, e.g. a human cell, e.g. a mouse cell.

Another aspect of the present invention features a polypeptide, of theCCR-protein family, which functions in one of either role of an agonistof cell-cycle regulation or an antagonist of cell-cycle regulation. In apreferred embodiment: the subject CCR-protein specifically binds acyclin dependent kinase (CDK), e.g. specifically binds CDK4; e.g.specifically binds CDK6; e.g. inhibits a kinase activity of CDK4;inhibits a kinase activity of CDK6; e.g. inhibits phosphorylation of anRB protein by CDK4. In a more preferred embodiment: the CCR-proteinregulates a eukaryotic cell-cycle, e.g. a mammalian cell-cycle, e.g., ahuman cell-cycle; the CCR-protein inhibits proliferation/cell growth ofa eukaryotic cell, e.g., a human cell; the CCR-protein inhibitsprogression of a eukaryotic cell from G₁ phase into S phase, e.g.,inhibits progression of a mammalian cell from G₁ phase into S phase.e.g., inhibits progression of a human cell from G₁ phase into S phase;the CCR-protein inhibits the kinase activity of a cyclin dependentkinase (CDK), e.g. a CDK active in G₁ phase, e.g. CDK 4; the CCR-proteinsuppresses tumor growth, e.g. in a tumor cell, e.g. in a tumor cellhaving an unimpaired RB or RB-like protein checkpoint. Moreover,CCR-proteins of the present invention may also have biologicalactivities which include: an ability to regulate cell-cycle progressionin response to extracellular factors and cytokines, e.g. functional inparacrine or autocrine regulation of cell growth and/or differentiation,e.g. inhibit CDK activation in response to transforming growth factor-β.(TGF-β) or related growth, differentiation or morphogenesis factor.

Yet another aspect of the present invention concerns an immunogencomprising a CCR-protein of the present invention, or a fragmentthereof, in an immunogenic preparation, the immunogen being capable ofeliciting an immune response specific for the CCR-protein; e.g. ahumoral response, eg. an antibody response; e.g. a cellular response.

Another aspect of the present invention features recombinantCCR-protein, or a fragment thereof, having an amino acid sequencepreferably: at least 60% homologous to the amino acid sequencerepresented in one of SEQ ID NO: 2, 4 or 6; at least 80% homologous tothe amino acid sequence represented in one of SEQ ID NO: 2, 4 or 6; atleast 90% homologous to the amino acid sequence represented in one ofSEQ ID NO: 2, 4 or 6; identical to the amino acid sequence representedin one of SEQ ID NO: 2, 4 or 6. In a preferred embodiment: the fragmentcomprises at least 5 contiguous amino acid residues of SEQ ID NO: 2, 4or 6; the fragment comprises at least 20 contiguous amino acid residuesof SEQ ID NO: 2, 4 or 6; the fragment comprises at least 50 contiguousamino acid residues of SEQ ID NO: 2, 4 or 6. In a preferred embodiment,the recombinant CCR-protein functions in one of either role of anagonist of cell-cycle regulation or an antagonist of cell-cycleregulation. In a more preferred embodiment: the CCR-protein specificallybinds a cyclin dependent kinase (CDK), e.g. specifically binds CDK4;e.g. specifically binds CDK6; e.g. inhibits a kinase activity of CDK4;inhibits a kinase activity of CDK6; e.g. inhibits phosphorylation of anRB protein by CDK4. In a more preferred embodiment: the CCR-proteinregulates a eukaryotic cell-cycle, e.g. a mammalian cell-cycle, e.g., ahuman cell-cycle; the CCR-protein inhibits proliferation/cell growth ofa eukaryotic cell, e.g., a human cell; the CCR-protein inhibitsprogression of a eukaryotic cell from G₁ phase into S phase, e.g.,inhibits progression of a mammalian cell from G₁ phase into S phase,e.g., inhibits progression of a human cell from G₁ phase into S phase;the CCR-protein inhibits the kinase activity of a cyclin dependentkinase (CDK), e.g. a CDK active in G₁ phase, e.g. CDK 4; the CCR-proteinsuppresses tumor growth, e.g. in a tumor cell, e.g. in a tumor cellhaving an unimpaired RB or RB-like protein checkpoint.

In yet other preferred embodiments, the recombinant CCR-protein is afusion protein further comprising a second polypeptide portion having anamino acid sequence from a protein unrelated the protein of SEQ ID NO:2, 4 or 6. Such fusion proteins can be functional in a two-hybrid assay.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a CCR-protein,or a fragment thereof, having an amino acid sequence at least 60%homologous to one of SEQ ID NOs: 2, 4 or 6. In a more preferredembodiment: the nucleic acid encodes a protein having an amino acidsequence at least 80% homologous to SEQ ID NO: 2, more preferably atleast 90% homologous to SEQ ID NO: 2, and most preferably at least 95%homologous to SEQ ID NO: 2; the nucleic acid encodes a protein having anamino acid sequence at least 80% homologous to SEQ ID NO: 6, morepreferably at least 90% homologous to SEQ ID NO: 6, and most preferablyat least 95% homologous to SEQ ID NO: 6. The nucleic preferably encodesa CCR-protein which specifically binds a cyclin dependent kinase (CDK);e.g. specifically binds CDK4; e.g. specifically binds CDK6; e.g. whichinhibits a kinase activity of CDK4; e.g. which inhibits phosphorylationof an RB protein by CDK4.

In another embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID NO: 1; more preferably to at least 20consecutive nucleotides of SEQ ID NO: 1; more preferably to at least 40consecutive nucleotides of SEQ ID NO: 1.

In a further embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID NO: 3; more preferably to at least 20consecutive nucleotides of SEQ ID NO: 3; more preferably to at least 40consecutive nucleotides of SEQ ID NO: 3.

In yet a further embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID NO: 5; more preferably to at least 20consecutive nucleotides of SEQ ID NO: 5; more preferably to at least 40consecutive nucleotides of SEQ ID NO: 5.

Furthermore, in certain embodiments, the CCR nucleic acid will comprisea transcriptional regulatory sequence, e.g. at least one of atranscriptional promoter or transcriptional enhancer sequence, operablylinked to the CCR-gene sequence so as to render the recombinant CCR-genesequence suitable for use as an expression vector.

The present invention also features transgenic non-human animals, e.g.mice, which either express a heterologous CCR-gene, e.g. derived fromhumans, or which mis-express their own CCR-gene, e.g. p16, p15 or p13.5expression is disrupted. Such a transgenic animal can serve as an animalmodel for studying cellular disorders comprising mutated ormis-expressed CCR alleles.

The present invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizesunder-stringent conditions to at least 10 consecutive nucleotides ofsense or antisense sequence of one of SEQ ID NO: 1, 3 or 5, or naturallyoccurring mutants thereof. In preferred embodiments, the probe/primerfurther comprises a label group attached thereto and able to bedetected, e.g. the label group is selected from a group consisting ofradioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.Such probes can be used as a part of a diagnostic test kit foridentifying transformed cells, such as for measuring a level of a p16,p15 or p13.5 encoding nucleic acid in a sample of cells isolated from apatient; e.g. for measuring the mRNA level in a cell or determiningwhether the genomic CCR gene has been mutated or deleted.

Probes may be labeled with any detectable group for use in practicingthe invention. Such detectable group can be any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and in general most anylabel useful in such methods can be applied to the present invention.Particularly useful are enzymatically active groups, such as enzymes(see Clin. Chem., 22:1243 (1976)), enzyme substrates (see British Pat.Spec. 1,548,741), coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565)and enzyme inhibitors (see U.S. Pat. No. 4,134,792); fluorescent markers(see Clin. Chem., 25:353 (1979); chromophores; luminescent compoundssuch as chemiluminescent and bioluminescent markers (see Clin. Chem.,25:512 (1979)); specifically bindable ligands; proximal interactingpairs; and radioisotopes such as ³H, ³⁵S, ³²P, ¹²⁵I and ¹⁴C.

In similar fashion, anti-p16 antibodies can be labeled and used todetect the presence of p16 protein in samples of cells.

In yet another embodiment, the present invention particularlycontemplates assays and kits for detecting p16 levels in cells.Antibodies specific for p16 (as described herein), or nucleic acidprobes directed to detecting mRNA levels of p16 transcripts, can be usedto detect transformed cells. As described above, the level of p16 mRNA,and presumably p16 protein, is elevated in transformed cells relative tonormal cells. Thus, detecting the level of p16 gene expression isdiagnostically useful in determining the presence of transformed cells.

The present invention also provides a method for treating an animalhaving unwanted cell growth characterized by a loss of wild-typeCCR-protein function, comprising administering a therapeuticallyeffective amount of an agent able to inhibit a kinase activity of a CDK,e.g. CDK4. In one embodiment, the method comprises administering anucleic acid construct encoding a CCR protein, e.g. p16, p15 or p13.5,e.g. a polypeptide represented in one of SEQ ID NOs: 2, 4 or 6, underconditions wherein the construct is incorporated by CCR-deficient cellsand the polypeptide is expressed, e.g. by gene therapy techniques. Inanother embodiment, the method comprises administering a CCR mimetic,e.g. a peptidomimetic, which binds to and inhibits the CDK.

Another aspect of the present invention provides a method of determiningif a subject, e.g. a human patient, is at risk for a disordercharacterized by unwanted cell proliferation, comprising detecting, in atissue of the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding aprotein represented by one of SEQ ID NOs: 2, 4 or 6, or a homologthereof; or (ii) the mis-expression of the CCR-gene, e.g. the p16, p15or p13.5 gene. In preferred embodiments: detecting the genetic lesioncomprises ascertaining the existence of at least one of a deletion ofone or more nucleotides from said gene, an addition of one or morenucleotides to said gene, an substitution of one or more nucleotides ofsaid gene, a gross chromosomal rearrangement of said gene, a grossalteration in the level of a messenger RNA transcript of said gene, thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of said gene, or a non-wild type level of said protein. Forexample, detecting the genetic lesion can comprise (i) providing aprobe/primer comprising an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequence ofone of SEQ ID NOs: 1, 3 or 5, or naturally occurring mutants thereof, or5′ or 3′ flanking sequences naturally associated with the CCR-gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and (iii)detecting, by hybridization of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion; e.g. wherein detecting thelesion comprises utilizing the probe/primer to determine the nucleotidesequence of the CCR-gene and, optionally, of the flanking nucleic acidsequences; e.g. wherein detecting the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR); e.g. whereindetecting the lesion comprises utilizing the probe/primer in a ligationchain reaction (LCR). In alternate embodiments, the level of saidprotein is detected in an immunoassay.

Yet another aspect of the invention pertains to a peptidomimetic whichbinds to a CCR-protein, e.g. p15 or p16, and inhibits its binding to aCDK, e.g. CDK4 or CDK6. For example, a preferred peptidomimetic is ananalog of a peptide having the sequenceVAEIG(V/E)GAYG(T/K)-V(F/Y)KARD-(SEQ ID NO: 15), though more preferablythe peptidomimetic is an analog of the hexa-peptide V(F/Y)KARD (SEQ IDNO: 16), and even more preferably of the tetrapeptide KARD (SEQ ID NO:17). Non-hydrolyzable peptide analogs of such residues can be generatedusing, for example, benzodiazepine, azepine, substituted gama lactamrings, keto-methylene pseudopeptides, β-turn dipeptide cores, orβ-aminoalcohols.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims. The practice of thepresent invention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a schematic representation of the p16 cDNA (SEQ ID NO:18), indicating the location of exon boundaries and PCR primers used inthe present invention.

FIGS. 2A and 2B are the genomic nucleic acid sequence for exon 1 (p16ex1(SEQ ID NO: 19), p16ex13 (SEQ ID NO: 20), p16 nt2 (SEQ ID NO: 21), p16nt3 (SEQ ID NO: 22)) and the non-coding sequences directly flankingexon 1. The sequence is a composite sequence from several primers. FIG.2C is the genomic sequence about exon 2 (p16int (SEQ ID NO: 23), p16ex15(SEQ ID NO: 24), p16ex2 (SEQ ID NO: 25), p16ex14 (SEQ ID NO: 26)).

FIGS. 3A-3D are the genomic nucleic acid sequence for exon 3 and thenon-coding sequences directly flanking exon 3 (p16EX5 (SEQ ID NO: 27),p16EX9 (SEQ ID NO: 28), p16EX4 (SEQ ID NO: 29), p16EX6 (SEQ ID NO: 30),p16EX6A (SEQ ID NO: 31)). The sequence is a composite sequence fromseveral primers.

FIG. 4 illustrates the loss of p16 sequences from the genomes of severalhuman tumor cells, as compared to normal human controls and other humantumors.

FIG. 5 illustrates the restriction map for the mouse p16 gene.

FIG. 6 is a sequence alignment of a highly conserved portion of theCCR-proteins p16 (SEQ ID NO: 32), p15 (SEQ ID NO: 33) and p13.5 (SEQ IDNO: 34).

DETAILED DESCRIPTION OF THE INVENTION

Progression through the cell-cycle is marked by a series of irreversibletransitions that separate discrete tasks necessary for faithful cellduplication. These transitions are negatively regulated by signals thatconstrain the cell-cycle until specific conditions are fulfilled. Entryin to mitosis, for example, is inhibited by incompletely replicated DNAor DNA damage. These restrictions on cell-cycle progression areessential for preserving the fidelity of the genetic information duringcell division. The transition from G.sub.1 to S phase, on the otherhand, coordinates cell proliferation with environmental cues, afterwhich the checks on the cell-cycle progression tend to be cellautonomous. Among the signals that restrict cell-cycle progressionduring G₁ are extracellular proteins which inhibit cell proliferation,growth factor or amino acid depletion, and cell-cell contact. Disruptionof these signalling pathways uncouples cellular responses fromenvironmental controls and may lead to unrestrained cell proliferation.

Eukaryotic cells, in general, require cyclin-dependent kinases (CDKs)for progression through G₁ and entry into S phase. In mammalian cells,both D- and E-type cyclins are rate limiting for the G₁ to S transition,and both reduce, but do not eliminate, the cell's requirement formitogenic growth factors. However, prior to the present discovery, therewas little information concerning the manner by which these cyclins andCDKs are negatively regulated by either intracellular or extracellularsignals that inhibit cell proliferation.

The present invention is directed to the discovery of a family ofrelated cell-cycle regulatory proteins (termed “CCR-proteins”) whichfunction typically to restrict progression of a cell through mitosis,and are likely to be involved in controlling progression throughmeiosis. Members of this family, apparently evolutionarily related,include a polypeptide (termed “p16”) having an apparent molecular weightof 16 Kd, another polypeptide (termed “p15”) having an approximatemolecular weight of 15 Kd, and a 13.5 Kd polypeptide (termed “p13.5”).The nucleotide sequences for the human p16, the human p15, and the mousep13.5 coding sequences are provided in SEQ ID NOs: 1, 3 and 5,respective, and a partial sequence for a mouse p16/p15 clone is providedin SEQ ID NO: 7. The corresponding amino acid sequences are representedin SEQ ID NOs: 2, 4, 6 and 8. An amino acid sequence for a partial humanp16 is represented in SEQ ID NO: 35. Moreover, data from hybridizationand immunoprecipitation experiments indicates still other members of theCCR-protein family exist.

One function of members of this family of proteins in cell-cycleregulation is in modulating the activity of cyclin/CDK complexes duringvarious stages of the cell-cycle, particularly those which include CDKsactive in G₁ phase, such as CDK4 or CDK6. To illustrate, both p16 andp15 are demonstrated below to exert an inhibitory effect on the activityof cyclin/CDK complexes, particularly those which include CDK4 or CDK6.For instance, each protein is able to inhibit the activity of cyclinD1/CDK complexes in vivo. As is generally known, cyclin D1 has beenassociated with a wide variety of proliferative diseases. Consequently,the present invention identifies a potential inhibitor of cellproliferation resulting from oncogenic expression of cyclin D1.Moreover, the diversity of members of the CCR-protein family, like thediversity of CDKs, is suggestive of individualistic roles of each memberof this family, which may be tissue-type of cell-type specific, occur atdifferent points in the cell-cycle, occur as part of differentextracellular or intracellular signalling pathways, or a combinationthereof.

As described in the examples below, certain of the CCR-proteins havebeen shown to be deleted or mutated at high frequency in tumors, such asderived from lung, breast, brain, bone, skin, bladder, kidney, ovary, orlymphocytes. Consequently, as set forth in the present application,replacement of CCR protein function by gene therapy or by CCR mimeties,or by direct inhibition of CDK4 or CDK6 activity, is therefore apotential therapy for treating such proliferative disorders. Moreover,the present data demonstrates that p15 expression is regulated bytreatment with transforming growth factor-β (TGF-β), suggesting that p15may function as an effector of TGF-β mediated cell cycle arrest viainhibition of CDK4 or CDK6 kinases. Considered in light of recentfindings demonstrating that reduced responsiveness to TGF-β may be animportant event in the loss of growth control in many proliferativedisorders, an approach to modulate CDK4/6 activity by CCR mimetics orCCR-gene therapy, or by mechanism based inhibitors of the kinasesthemselves, is even more attractive for treating such proliferativedisorders.

Accordingly, the present invention makes available diagnostic andtherapeutic assays and reagents for detecting and treating proliferativedisorders arising from, for example, tumorigenic transformation ofcells, or other hyperplastic or neoplastic transformation processes, aswell as differentiative disorders, such as degeneration of tissue, e.g.neurodegeneration. For example, the present invention makes availablereagents, such as antibodies and nucleic acid probes, for detectingaltered complex formation, and/or altered levels of CCR-proteinexpression, and/or CCR-gene deletion or mutation, in order to identifytransformed cells. Moreover, the present invention provides a method oftreating a wide variety of pathological cell proliferative conditions,such as by gene therapy utilizing recombinant gene constructs encodingthe subject CCR-proteins, or by providing CCR-mimetics, with the generalstrategy being the inhibition of adherently proliferating cells.

The subject proteins can also be used in assay systems to identifyagents which either decrease the ability of the CCR-protein to bind aCDK (e.g. CDK4 or CDK6) and thereby relieve inhibition of cyclin/CDKcomplexes, or alternatively, which agonize or mimic the CCR-mediatedinhibition of CDK activation. In the latter, e.g. CCR mimetics, theconsequence of inhibiting activation of a cyclin/CDK complex, e.g.cyclin D/CDK4, is the failure of the cell to advance through thecell-cycle, which inhibition can lead ultimately to cell death.Reactivation of the CDK/cyclin complexes, on the otherhand, can disruptor otherwise unbalance the cellular events occurring in a transformedcell. Such agents can be of use therapeutically to activate CDK4complexes in cells transformed, for example, by tumor viruses. Treatmentof such cells can cause premature progression through a checkpoint, e.g.the retinoblastoma (RB) checkpoint, and result in mitotic catastrophe(cell death) or induction of apoptosis.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the terms “gene”, “recombinant gene” and “geneconstruct” refer to a nucleic acid comprising an open reading frameencoding a cell-cycle regulatory of the present invention, includingboth exon and (optionally) intron sequences. In preferred embodiments,the nucleic acid is DNA or RNA. Exemplary recombinant genes includenucleic acids which encode all or a CDK-binding portion of the p16protein represented in SEQ ID NO: 2, the p15 protein represented in SEQID NO: 4, or the p13.5 protein represented in SEQ ID NO: 6. The term“intron” refers to a DNA sequence present in a given CCR-gene which isnot translated into protein and is generally found between exons.

“Homology” refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Homology can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

The term “transfection” refers to the introduction of a nucleic acid,e.g., an expression vector, into a recipient cell by nucleicacid-mediated gene transfer. “Transformation”, as used herein, refers toa process in which a cell's genotype is changed as a result of thecellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of one of the subjectcell-cycle regulatory proteins, e.g. p16, p15 or p13.5.

“Cells” or “cell cultures” or “recombinant host cells” or “host cells”are often used interchangeably as will be clear from the context. Theseterms include the immediate subject cell which expresses the cell-cycleregulatory protein of the present invention, and, of course, the progenythereof. It is understood that not all progeny are exactly identical tothe parental cell, due to chance mutations or difference in environment.However, such altered progeny are included in these terms, so long asthe progeny retain the characteristics relevant to those conferred onthe originally transformed cell. In the present case, such acharacteristic might be the ability to produce a recombinantCCR-protein.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. The term “expression vector” includes plasmids, cosmids orphages capable of synthesizing the subject CCR-protein encoded by therespective recombinant gene carried by the vector. Preferred vectors arethose capable of autonomous replication and/expression of nucleic acidsto which they are linked. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. Moreover, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, as well as polyadenylation sites, which induceor control transcription of protein coding sequences with which they areoperably linked. In preferred embodiments, transcription of arecombinant CCR-gene is under the control of a promoter sequence (orother transcriptional regulatory sequence) which controls the expressionof the recombinant gene in a cell-type in which expression is intended.It will also be understood that the recombinant gene can be under thecontrol of transcriptional regulatory sequences which are the same orwhich are different from those sequences which control transcription ofthe naturally-occurring form of the regulatory protein.

The term “tissue-specific promoter” means a DNA sequence that serves asa promoter, i.e., regulates expression of a selected DNA sequenceoperably linked to the promoter, and which effects expression of theselected DNA sequence in specific cells of a tissue, such as cells of aneuronal lineage, e.g. glial cells, or alternatively, in epithelialcells, e.g. melanocytes. In an illustrative embodiment, gene constructsutilizing glial-specific promoters can be used as a part of gene therapyto cause expression of recombinant forms of one of the subjectCCR-proteins in glioma cells with a feature of the gene construct beinga tissue-specific promoter for directing expression of the subjectprotein in only glial tissue. The term also covers so-called “leaky”promoters, which regulate expression of a selected DNA primarily in onetissue, but cause expression in other tissues as well.

As used herein, a “transgenic animal” is any animal, preferably anon-human mammal in which one or more of the cells of the animal containheterologous nucleic acid introduced by way of human intervention, suchas by transgenic techniques well known in the art. The nucleic acid isintroduced into the cell, directly or indirectly by introduction into aprecursor of the cell, by way of deliberate genetic manipulation, suchas by microinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classical cross-breeding, or invitro fertilization, but rather is directed to the introduction of arecombinant DNA molecule. This molecule may be integrated within achromosome, or it may be extrachromosomally replicating DNA. In thetypical transgenic animals described herein, the transgene causes cellsto express a recombinant form of the subject CCR proteins, e.g. eitheragonistic or antagonistic forms, or in which an endogenous CCR-gene hasbeen disrupted. However, transgenic animals in which the recombinantCCR-gene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. The “non-humananimals” of the invention include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, amphibians, reptiles, etc. Preferrednon-human animals are selected from the rodent family including rat andmouse, most preferably mouse. The term “chimeric animal” is used hereinto refer to animals in which the recombinant gene is found, or in whichthe recombinant is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that therecombinant CCR-gene is present and/or expressed, or disrupted, in sometissues but not others.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., a CCR-polypeptide), which is partly or entirelyheterologous, i.e., foreign, to the transgenic animal or cell into whichit is introduced, or, is homologous to an endogenous gene of thetransgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

As used herein, the terms “transforming growth factor-β” and “TGF-β”denote a family of structurally related paracrine polypeptides foundubiquitously in vertebrates, and prototypic of a large family ofmetazoan growth, differentiation, and morphogenesis factors (see, forreview, Massaque et al. (1990) Ann Rev Cell Biol 6:597-641; and Spom etal. (1992) J Cell Biol 119:1017-1021).

The term “evolutionarily related to”, with respect to nucleic acidsequences encoding CCR-proteins, refers to nucleic-acid-sequences whichhave arisen naturally in an organism, including naturally occurringmutants. The term also refers to nucleic acid sequences which, whilederived from a naturally occurring CCR-proteins, have been altered bymutagenesis, as for example, combinatorial mutagenesis described below,yet still encode polypeptides which have at least one activity of aCCR-protein. For instance, the sequence of p16 can be altered bymutagenesis based on amino acid substitutions derived from alignmentwith the p15 and/or p13.5 sequences.

One aspect of the present invention pertains to an isolated nucleic acidcomprising the nucleotide sequence encoding a CCR-protein, fragmentsthereof encoding polypeptides having at least one biological activity ofa CCR-protein, and/or equivalents of such nucleic acids. The termnucleic acid as used herein is intended to include such fragments andequivalents. The term equivalent is understood to include nucleotidesequences encoding functionally equivalent CCR-proteins or functionallyequivalent peptides having an activity of a CCR-protein such asdescribed herein. Equivalent nucleotide sequences will include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will also include sequencesthat differ from the nucleotide sequence encoding the subject p16protein represented by SEQ ID NO: 2, or the p15 protein represented bySEQ ID NO: 4, or the p13.5 protein represented by SEQ ID NO: 6 due tothe degeneracy of the genetic code. Equivalents will also includenucleotide sequences that hybridize under stringent conditions (i.e.,equivalent to about 20-27° C. below the melting temperature (T_(m)) ofthe DNA duplex formed in about 1M salt) to the nucleotide sequence of aCCR-gene shown in SEQ ID NO: 1, 3 or 5. In one embodiment, equivalentswill further include nucleic acid sequences derived from andevolutionarily related to the nucleotide sequence shown in one of SEQ IDNO: 1, 3 or 5.

The term “isolated” as also used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding on of thesubject CCR-proteins preferably includes no more than 10 kilobases (kb)of nucleic acid sequence which naturally immediately flanks the CCR-genein genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized. Moreover, an “isolated nucleic acid” is meant toinclude nucleic acid fragments which are not naturally occurring asfragments and would not be found in the natural state.

Polypeptides referred to herein as having an activity of a cell-cycleregulatory protein preferably have an amino acid sequence correspondingto all or a portion of the amino acid sequence of the p16 protein shownin SEQ ID NO: 2, of the p15 protein shown in SEQ ID NO: 4, or of thep13.5 protein shown in SEQ ID NO: 6, or isoforms of one of theseproteins (including differential splicing variants). In preferredembodiments, the biological activity of a CCR-protein includes: anability to regulate a eukaryotic cell-cycle, e.g. a mammaliancell-cycle, e.g., a human cell-cycle; an ability to inhibitproliferation/cell growth of a eukaryotic cell, e.g. a mammalian cell,e.g., a human cell; an ability to inhibit progression of a eukaryoticcell from G₁ phase into S phase, e.g., inhibit progression of amammalian cell from G₁ phase into S phase, e.g., inhibit progression ofa human cell from G₁ phase into S phase; an ability to inhibit thekinase activity of a cyclin dependent kinase (CDK), e.g. a CDK active inG₁ phase, e.g. CDK 4, e.g. CDK6; e.g. an ability to inhibitphosphorylation of a retinoblastoma (RB) or retinoblastoma-like proteinby a cyclin dependent kinase. Moreover, CCR-proteins of the presentinvention may also have biological activities which include: an abilityto suppress tumor growth, e.g. in a tumor having an unimpaired RBprotein; an ability to regulate cell-cycle progression in response toextracellular factors and cytokines, e.g. functional in paracrine orautocrine regulation of cell growth and/or differentiation, e.g. inhibitCDK activation in response to transforming growth factors (TGF-β) orrelated growth, differentiation or morphogenesis factor. In thisrespect, the CCR-proteins of the present invention may also function toprevent de-differentiation of cells/tissue. Other biological activitiesof the subject CCR-proteins are described herein or will be reasonablyapparent to those skilled in the art in light of the present disclosure.

Moreover, it will be generally appreciated that, under certaincircumstances, it will be advantageous to provide homologs ofnaturally-occurring forms of particular CCR-proteins which are eitheragonists or antagonists of only a subset of that protein's biologicalactivities. Thus, specific biological effects can be elicited bytreatment with a homolog of limited function, and with fewer sideeffects relative to treatment with agonists or antagonists which aredirected to all of the biological activities of that protein. Forexample, p16 homologs can be generated which bind to and inhibitactivation of CDK4 without substantially interfering with the activationof CDK6.

In one embodiment, the nucleic acid of the invention encodes a peptidewhich is an agonist or antagonist of the p16 protein and comprises anamino acid sequence shown in SEQ ID NO: 2. Preferred nucleic acidsencode a peptide having a p16 protein activity and being at least 60%homologous, more preferably 70% homologous and most preferably 80%homologous with an amino acid sequence shown in SEQ ID NO: 2. Nucleicacids which encode peptides having an activity of a p16 protein andhaving at least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with a sequence shown in SEQID NO: 2 are also within the scope of the invention. Preferably, thenucleic acid is a cDNA molecule comprising at least a portion of thenucleotide sequence encoding a p16 protein shown in SEQ ID NO: 1. Apreferred portion of the cDNA molecule shown in SEQ ID NO: 1 includesthe coding region of the molecule.

In another embodiment, the nucleic acid of the invention encodes apeptide which is an agonist or antagonist of the p15 protein andcomprises an amino acid sequence shown in SEQ ID NO: 4. Preferrednucleic acids encode a peptide having a p15 protein activity and beingat least 60% homologous, more preferably 70% homologous and mostpreferably 80% homologous with an amino acid sequence shown in SEQ IDNO: 4. Nucleic acids which encode peptides having an activity of a p15protein and having at least about 90%, more preferably at least about95%, and most preferably at least about 98-99% homology with a sequenceshown in SEQ ID NO: 4 are also within the scope of the invention. In arepresentative embodiment, the nucleic acid is a cDNA moleculecomprising at least a portion of the nucleotide sequence encoding a p15protein shown in SEQ ID NO: 3. A preferred portion of the cDNA moleculeshown in SEQ ID NO: 3 includes the coding region of the molecule.

In yet another embodiment, the nucleic acid of the invention encodes apeptide having an activity of a p13.5 protein and comprising an aminoacid sequence shown in SEQ ID NO: 6. Preferred nucleic acids encode apeptide having a p13.5 protein activity and being at least 60%homologous, more preferably 70% homologous and most preferably 80%homologous with an amino acid sequence shown in SEQ ID NO: 6. Nucleicacids which encode peptides having an activity of a p13.5 protein, suchas the ability to bind a CDK, and having at least about 90%, morepreferably at least about 95%, and most preferably at least about 98-99%homology with a sequence shown in SEQ ID NO: 6 are also within the scopeof the invention. Preferably, the nucleic acid is a cDNA moleculecomprising at least a portion of the nucleotide sequence encoding ap13.5 protein shown in SEQ ID NO: 5. A preferred portion of the cDNAmolecule shown in SEQ ID NO: 5 includes the coding region of themolecule.

In yet another embodiment, the nucleic acid of the invention encodes apeptide having an amino acid sequence shown in SEQ ID NO: 8. Preferrednucleic acids encode a peptide having a CCR-protein activity and beingat least 60% homologous, more preferably 70% homologous and mostpreferably 80% homologous with an amino acid sequence shown in SEQ IDNO: 8. Nucleic acids which encode peptides having an activity of aCCR-protein, such as the ability to bind a CDK, and having at leastabout 90%, more preferably at least about 95%, and most preferably atleast about 98-99% homology with a sequence shown in SEQ ID NO: 8 arealso within the scope of the invention. Preferably, the nucleic acid isa cDNA molecule comprising the nucleotide sequence shown in SEQ ID NO:7.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to a nucleic acid which encodesa CCR polypeptide having all or a portion of an amino acid sequenceshown in one of SEQ ID NOs: 2, 4, 6 or 8. Appropriate stringencyconditions which promote DNA hybridization, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50 C, are known to those skilled in the art cr can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. For example, the salt concentration in the washstep can be selected from a low stringency of about 2.0×SSC at 50° C. toa high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C.

Isolated nucleic acids which differ from the nucleotide sequences shownin one of SEQ ID NOs: 1, 3, 5 or 7 due to degeneracy in the genetic codeare also within the scope of the invention. For example, a number ofamino acids are designated by more than one triplet. Codons that specifythe same amino acid, or synonyms (for example, CAU and CAC are synonymsfor histidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject CCR-proteins will exist among eukaryotic cells.One skilled in the art will appreciate that these variations in one ormore nucleotides (up to about 3-4% of the nucleotides) of the nucleicacids encoding a particular member of the CCR-protein family may existamong individuals of a given species due to natural allelic variation.Any and all such nucleotide variations and resulting amino acidpolymorphisms are within the scope of this invention.

Fragments of the nucleic acid encoding a biologically active portion ofthe subject CCR-proteins are also within the scope of the invention. Asused herein, a fragment of the nucleic acid encoding an active portionof a CCR-protein refers to a nucleotide sequence having fewernucleotides than the nucleotide sequence encoding the full length aminoacid sequence of, for example, the CCR-proteins represented in SEQ IDNOs: 2, 4 or 6, and which encodes a peptide which retains at least aportion of the biological activity of the full-length protein (i.e., apeptide capable of binding a CDK) as defined herein, or alternatively,which is functional as an antagonist of the biological activity of thefull-length protein. Nucleic acid fragments within the scope of theinvention include those capable of hybridizing under high or lowstringency conditions with nucleic acids from other species, e.g. foruse in screening protocols to detect homologs of the subjectCCR-proteins. Nucleic acids within the scope of the invention may alsocontain linker sequences, modified restriction endonuclease sites andother sequences useful for molecular cloning, expression or purificationof such recombinant peptides.

As indicated by the examples set out below, a nucleic acid encoding apeptide having an activity of a CCR-protein may be obtained from mRNA orgenomic DNA present in any of a number of eukaryotic cells in accordancewith protocols described herein, as well as those generally known tothose skilled in the art. A cDNA encoding a CCR-protein, for example,can be obtained by isolating total mRNA from a cell, e.g. a mammaliancell, e.g. a human cell. Double stranded cDNAs can then be prepared fromthe total mRNA, and subsequently inserted into a suitable plasmid orbacteriophage vector using any one of a number of known techniques. Agene encoding a CCR-protein can also be cloned using establishedpolymerase chain reaction techniques in accordance with the nucleotidesequence information provided by the invention. A preferred nucleic acidis a cDNA encoding a p16 protein having a sequence shown in SEQ IDNO: 1. Another preferred nucleic acid is a cDNA encoding a p15 proteinhaving a sequence shown in SEQ ID NO: 3. Yet another preferred nucleicacid is a cDNA encoding a p13.5 protein having a sequence shown in SEQID NO: 5.

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotide probesor their derivatives which specifically hybridizes (e.g. binds) undercellular conditions, with the cellular mRNA and/or genomic DNA encodinga CCR-protein so as to inhibit expression of that protein, e.g. byinhibiting transcription and/or translation. The binding may be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interactions in the majorgroove of the double helix. In general, “antisense” therapy refers tothe range of techniques generally employed in the art, and includes anytherapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a CCR-protein. Alternatively, the antisenseconstruct is an oligonucleotide probe which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA and/or genomic sequences encoding one of thesubject CCR proteins. Such oligonucleotide probes are preferablymodified oligonucleotide which are resistant to endogenous nucleases,e.g. exonucleases and/or endonucleases, and is therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by van der krol etal. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous for injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

This invention also provides expression vectors comprising a nucleotidesequence encoding a subject cell-cycle regulatory protein and operablylinked to at least one regulatory sequence. Operably linked is intendedto mean that the nucleotide sequence is linked to a regulatory sequencein a manner which allows expression of the nucleotide sequence.Regulatory sequences are art-recognized and are selected to directexpression of the peptide having an activity of a CCR-protein.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). For instance,any of a wide variety of expression control sequences-sequences thatcontrol the expression of a DNA sequence when operatively linked to itmay be used in these vectors to express DNA sequences encoding theCCR-proteins of this invention. Such useful expression controlsequences, include, for example, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage lambda, the control regions for fd coat protein, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, the promoters of the yeast.alpha.-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered.

As will be apparent, the subject gene constructs can be used to causeexpression of the subject CCR-proteins in cells propagated in culture,e.g. to produce proteins or peptides, including fusion proteins orpeptides, for purification. In addition, recombinant expression of thesubject CCR-proteins, or agonist forms thereof, in cultured cells can beuseful for preventing de-differentiation of cells in vitro. Toillustrate, in vitro neuronal culture systems have proved to befundamental and indispensable tools for the study of neural development,as well as the identification of neurotrophic factors. Once a neuronalcell has become terminally-differentiated, it typically will not changeto another terminally differentiated cell-type. However, neuronal cellscan nevertheless readily lose their differentiated state. This iscommonly observed when they are grown in culture from adult tissue, andwhen they form a blastoma during regeneration.

Agonizing the function of the subject CCR-proteins, such as bymaintaining expression (or overexpression) of p16 or p15, provides ameans for ensuring an adequately restrictive environment in order tomaintain neuronal cells at various stages of differentiation, and can beemployed, for instance, in cell cultures designed to test the specificactivities of trophic factors. Other tissue culture systems whichrequire maintenance of differentiation will be readily apparent to thoseskilled in the art. In this respect, each of the agonist and antagonistof CCR inhibition of CDK4 and CDK6 can be used for ex vivo tissuegeneration, as for example, to enhance the generation of prostheticcartilage devices for implantation.

Conversely, by antagonizing the activity of the wild-type CCR-proteins,such as by expression of antagonistic homologs, antisense constructs, ortreatment with agents able to disrupt binding of a CCR-protein with aCDK, the cultured cells can be prevented from following certaindifferentiative pathways, and, importantly, can cause transformation ofcells in culture. In similar fashion, the dominant negative CDK4 andCDK6 mutants described below can be used to cause cellulartransformation, as each of these CDK mutants is insensitive to p16inhibition and is effectively an antagonist of p16. The ability of CCRantagonists to promote cell growth is particularly significant in lightof the observation that human cells are notoriously difficult to grow invitro. Accordingly, such reagents are therefore useful for transforming,and in certain instances, immortalizing, cells from primary cellcultures.

Moreover, the subject gene constructs can also be utilized in diagnosticassays to determine if a cell's growth is no longer dependent on theregulatory function of a CCR-protein, e.g. in determining the phenotypeof a transformed cell. To illustrate, a sample of cells from the tissuecan be obtained from a patient and dispersed in appropriate cell culturemedia, a portion of the cells in the sample can be caused to express arecombinant CCR-protein, e.g. by transfection with a p16, p15 or p13.5expression vector, and subsequent growth of the cells assessed. Theability of cells to proliferate despite expression of the CCR-protein isindicative of a lack of dependence on cell regulatory pathways whichinclude the CCR-protein, e.g. RB-mediated checkpoints. Depending on thenature of the tissue of interest, the sample can be in the form of cellsisolated from, for example, a blood sample, an exfoliated cell sample, afine needle aspirant sample, or a biopsied tissue sample. Where theinitial sample is a solid mass, the tissue sample can be minced orotherwise dispersed so that cells can be cultured, as is known in theart. Such knowledge can have both prognostic and therapeutic benefits.

This invention also pertains to a host cell transfected with arecombinant CCR-gene in order to express a polypeptide having anactivity of a CCR-protein. The host cell may be any prokaryotic oreukaryotic cell. For example, a CCR-protein of the present invention maybe expressed in bacterial cells such as E. coli, insect cells(baculovirus), yeast, or mammalian cells. Other suitable host cells areknown to those skilled in the art.

Another aspect of the present invention concerns recombinantCCR-proteins which are encoded by genes derived from eukaryoticorganisms, e.g. mammals, e.g. humans, and which have at least onebiological activity of a CCR-protein, or which are naturally occurringmutants thereof. The term “recombinant protein” refers to a protein ofthe present invention which is produced by recombinant DNA techniques,wherein generally DNA encoding the CCR-protein is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant gene encoding the recombinantCCR-protein, is meant to include within the meaning of “recombinantprotein” those proteins having an amino acid sequence of a nativeCCR-protein, or an amino acid sequence similar thereto which isgenerated by mutations including substitutions and deletions of anaturally occurring CCR-protein of a organism. To illustrate,recombinant proteins preferred by the present invention, in addition tonative p16, p15 or p13.5 proteins, are those recombinantly producedproteins which are at least 60% homologous, more preferably 70%homologous and most preferably 80% homologous with an amino acidsequence shown in SEQ ID NO: 2, 4, 6 or 8. Polypeptides having anactivity of a CCR-protein, such as CDK-binding, and having at leastabout 90%, more preferably at least about 95%, and most preferably atleast about 98-99% homology with a sequence shown in SEQ ID NO: 2, 4, 6or 8 are also within the scope of the invention. Thus, the presentinvention pertains to recombinant CCR-proteins which are encoded bygenes derived from a organism and which have amino acid sequencesevolutionarily related to a CCR-protein represented by one of ID No. 2,4 or 6, wherein “evolutionarily related to”, refers to CCR-proteinshaving amino acid sequences which have arisen naturally (e.g. by allelicvariance or by differential splicing), as well as mutational variants ofCCR-proteins which are derived, for example, by combinatorialmutagenesis.

The present invention further pertains to methods of producing thesubject CCR-proteins. For example, a host cell transfected withexpression vector encoding one of the subject CCR-protein can becultured under appropriate conditions to allow expression of the peptideto occur. The peptide may be secreted and isolated from a mixture ofcells and medium containing the peptide. Alternatively, the peptide maybe retained cytoplasmically and the cells harvested, lysed and theprotein isolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The peptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for particular epitopes of the subject CCR-proteins.In a preferred embodiment, the CCR-protein is a fusion proteincontaining a domain which facilitates its purification, such as p16-GST,p15-GST, or a p13.5-GST fusion proteins.

Thus, a nucleotide sequence derived from the cloning of a CCR-protein ofthe present invention, encoding all or a selected portion of theprotein, can be used to produce a recombinant form of the protein viamicrobial or eukaryotic cellular processes. Ligating the polynucleotidesequence into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial cells), arestandard procedures used in producing other well-known proteins, e.g.insulin, interferons, human growth hormone, IL-1, IL-2, and the like.Similar procedures, or modifications thereof, can be employed to preparerecombinant CCR-proteins, or portions thereof, by microbial means ortissue-culture technology in accord with the subject invention.

The recombinant CCR-protein can be produced by ligating the cloned gene,or a portion thereof, into a vector suitable for expression in eitherprokaryotic cells, eukaryotic cells, or both. Expression vehicles forproduction of a recombinant CCR-protein include plasmids and othervectors. For instance, suitable vectors for the expression of p16, p15or p13.5 include plasmids of the types: pBR322-derived plasmids,pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids andpUC-derived plasmids for expression in prokaryotic cells, such as E.coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example. Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

The preferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Examplesof other viral (including retroviral) expression systems can be foundbelow in the description of gene therapy delivery systems. The variousmethods employed in the preparation of the plasmids and transformationof host organisms are well known in the art. For other suitableexpression systems for both prokaryotic and eukaryotic cells, as well asgeneral recombinant procedures, see Molecular Cloning A LaboratoryManual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press: 1989) Chapters 16 and 17. In some instances, itmay be desirable to express the recombinant CCR-protein by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBLUEBAC®-derivedvectors (such as the β-gal containing pBLUEBAC® III).

When expression of a carboxy terminal fragment of the full-lengthCCR-protein is desired, i.e. a truncation mutant, it may be necessary toadd a start codon (ATG) to the oligonucleotide fragment containing thedesired sequence to be expressed. It is well known in the art that amethionine at the N-terminal position can be enzymatically cleaved bythe use of the enzyme methionine aminopeptidase (MAP). MAP has beencloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757)and Salmonella typhimurium and its in vitro activity has beendemonstrated on recombinant proteins (Miller et al. (1987) PNAS84:2718-1722). Therefore, removal of an N-terminal methionine, ifdesired, can be achieved either in vivo by expressing such recombinantpolypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP (e.g., procedure ofMiller et al.).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of one of the subject CCR-proteins. For example, the VP6 capsidprotein of rotavirus can be used as an immunologic carrier protein forportions of polypeptide, either in the monomeric form or in the form ofa viral particle. The nucleic acid sequences corresponding to theportion of the CCR protein to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising a portionof the protein as part of the virion. The Hepatitis B surface antigencan also be utilized in this role as well. Similarly, chimericconstructs coding for fusion proteins containing a portion of aCCR-protein and the poliovirus capsid protein can be created to enhanceimmunogenicity (see, for example, EP Publication No. 0259149; and Evanset al. (1989) Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; andSchlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canbe utilized, wherein a desired portion of a CCR-protein is obtaineddirectly from organo-chemical synthesis of the peptide onto anoligomeric branching lysine core (see, for example, Posnett et al.(1988) JBC 263:1719 and Nardelli et al. (1992) J. Immunol. 148:914).Antigenic determinants of the CCR-proteins can also be expressed andpresented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins. For example, the CCR-protein of the presentinvention can be generated as a glutathione-S-transferase (GST) fusionproteins. Such GST fusion proteins can be used to simply purification ofthe CCR-protein, such as through the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausabel et al. (N.Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified CCR-protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992).

The present invention also makes available isolated and/or purifiedforms of the subject CCR-proteins, which are isolated from, or otherwisesubstantially free of other extracellular proteins, especiallycell-cycle proteins, e.g. CDKs, cyclins, p21, p19, or PCNA, normallyassociated with the CCR-protein. The term “substantially free of othercellular proteins” (also referred to herein as “contaminating proteins”)is defined as encompassing, for example, p16, p15 or p13.5 preparationscomprising less than 20% (by dry weight) contaminating protein, andpreferably comprises less than 5% contaminating protein. Functionalforms of the CCR-proteins can be prepared, for the first time, aspurified preparations by using a cloned gene as described herein. By“purified”, it is meant, when referring to a polypeptide, that theindicated molecule is present in the substantial absence of otherbiological macromolecules, such as other proteins (particularly othercycle proteins such as CDK4 or CDK6, as well as other contaminatingproteins). The term “purified” as used herein preferably means at least80% by dry weight, more preferably in the range of 95-99% by weight, andmost preferably at least 99.8% by weight, of biological macromoleculesof the same type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above. “Isolated” and“purified” do not encompass either natural materials in their nativestate or natural materials that have been separated into components(e.g., in an acrylamide gel) but not obtained either as pure (e.g.lacking contaminating proteins, or chromatography reagents such asdenaturing agents and polymers, e.g. acrylamide or agarose) substancesor solutions.

Moreover, isolated peptidyl portions of the subject CCR-proteins canalso be obtained by screening peptides recombinantly produced from thecorresponding fragment of the nucleic acid encoding such peptides. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a CCR-protein of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of, for example,CDK4 activation, such as by microinjection assays. In an illustrativeembodiment, peptidyl portions of the subject CCR proteins can tested forCDK-binding activity, as well as inhibitory ability, by expression as,for example, thioredoxin fusion proteins each of which contains adiscrete fragment of the CCR-protein (see, for example, U.S. Pat. Nos.5,270,181 and 5,292,646; and PCT publication WO94/02502).

It is also possible to modify the structure of the subject CCR-proteinsfor such purposes as enhancing therapeutic or prophylactic efficacy, orstability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified polypeptides, when designed toretain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the cell-cycleregulatory proteins described in more detail herein. Such modifiedpolypeptides can be produced, for instance, by amino acid substitution,deletion, or addition.

Moreover, it is reasonable to expect, for example, that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. conservativemutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In similar fashion, the amino acid repertoirecan be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine. (see, for example,Biochemistry, 2nd ed, Ed. by L. Stryer, WH Freeman and Co.: 1981).Whether a change in the amino acid sequence of a peptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant peptide to produce a response in cells in a fashion similarto the wild-type protein. For instance, such variant forms of p16 can beassessed for their ability to complement a p16-deficient cell. Peptidesin which more than one replacement has taken place can readily be testedin the same manner.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the present CCR-proteins, as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g. homologs) that are functional in binding to a CDK,especially CDK4 or CDK6. The purpose of screening such combinatoriallibraries is to generate, for example, novel p16, p15 or p13.5 homologswhich can act as either agonists or antagonist, or alternatively,possess novel activities all together. To illustrate, p16 and/or p15homologs can be engineered by the present method to provide moreefficient binding to CDK4, yet have a significantly reduced bindingaffinity for CDK6 relative to the naturally-occurring form of theprotein. Thus, combinatorially-derived homologs can be generated whichhave a selective potency relative to a naturally occurring CCR-protein.Such proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols.

Likewise, mutagenesis can give rise to CCR homologs which haveintracellular half-lives dramatically different than the correspondingwild-type protein. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or othercellular process which result in destruction of, or otherwiseinactivation of, the CCR-protein. Such homologs, and the genes whichencode them, can be utilized to alter the envelope of p16, p15 or p13.5expression by modulating the half-life of the protein. For instance, ashort half-life can give rise to more transient biological effects and,when part of an inducible expression system, can allow tighter controlof recombinant CCR-protein levels within the cell. As above, suchproteins, and particularly their recombinant nucleic acid constructs,can be used in gene therapy protocols.

In similar fashion, CCR homologs can be generated by the presentcombinatorial approach to act as antagonists, in that they are able tointerfere with the ability of the corresponding wild-type protein toregulate cell proliferation.

In a representative embodiment of this method, the amino acid sequencesfor a population of CCR-protein homologs are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, p16 and/or p15 homologs from one or more species,or homologs from the same species but which differ due to mutation.Amino acids which appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences. Thepresence or absence of amino acids from an aligned sequence of aparticular variant is relative to a chosen consensus length of areference sequence, which can be real or artificial. In order tomaintain the highest homology in alignment of sequences, deletions inthe sequence of a variant relative to the reference sequence can berepresented by an amino acid space (*), while insertional mutations inthe variant relative to the reference sequence can be disregarded andleft out of the sequence of the variant when aligned.

To illustrate, upon inspection of the p16, p15 and p13.5 sequences (seeFIG. 6), it was noted that an internal fragment of the threeCCR-proteins was highly conserved. Based on these alignments,combinatorial libraries can be generated from this portion, the membersof which can be expressed in the absence of other portions of aCCR-protein, or as a part of a CCR-protein in which other portions ofthe protein are static (e.g. a p16 or p15 protein in which only residuesMet-52 to Gly-135 or Met-53 to Gly-136, respectively, are varied bycombinatorial mutagenesis). For instance, a library of p16 or p15proteins comprise an amino acid sequence represented by the degenerateamino acid sequence:

(SEQ ID NO: 11) Met-Met-Met-Gly-Xaa(1)-Xaa(2)-Xaa(3)-Val-Ala-Xaa(4)-Leu-Leu-Leu- Xaa(5)-Xaa(6)-Gly-Ala-Xaa(7)-Xaa(8)-Asn-Cys-Xaa(9)-Asp-Pro-Xaa(10)- Thr-Xaa(11)-Xaa(12)-Xaa(13)-Arg-Pro-Val-His-Asp- -Ala- Ala-Arg-Glu-Gly-Phe-Leu-Asp-Thr-Leu-Val-Val-Leu- His--Xaa(14)-Xaa(15)-Gly-Ala-Arg-Leu-Asp-Val-Arg- Asp-Ala-Trp-G- ly-Arg-Leu-Pro-Xaa(16)-Asp-Leu-Ala- Xaa(17)-Glu-Xaa(18)-Gly-His-Xaa(19)-Asp-Xaa(20)- Xaa(21)-Xaa(22)-Tyr-Leu-Arg-Xaa(23)-Ala-Xaa(24)- Gly

wherein each of Xaa(1)-Xaa(24) is selected from one of the amino acidresidues of the same position in SEQ ID NO: 2, 4 or 6.

Further expansion of the combinatorial library can be made by, forexample, by including amino acids which would represent conservativemutations at one or more of the degenerate positions. Inclusion of suchconservative mutations can give rise to a library of potentialcell-cycle regulatory sequences represented by the above formula, butwherein Xaa(1) represents Ser, Thr, Asn or Gln; Xaa(2) represents Gly,Ala, Val, Leu, or Ile; Xaa(3) represents Arg, Lys or His; Xaa(4)represents Gly, Ala, Val, Leu, Ile, Asp or Glu; Xaa(5) represents Gly,Ala, Val, Leu, Ile, Asn or Gln; Xaa(6) represents Arg, Lys, His, Tyr orPhe; Xaa(7) represents Asp or Glu; Xaa(8) represents Pro, Gly, Ser orThr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Asp or Glu; Xaa(10)represents Gly, Ala, Val, Leu, Ile, or an amino acid gap; Xaa(11)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(12) represents Phe,Tyr, Trp or an amino acid gap; Xaa(13) represents Ser or Thr; Xaa(14)represents Gly, Ala, Val, Leu, Ile, Arg, Lys or H is; Xaa(15) representsGly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(16) represents Gly, Ala, Val,Leu or Ile; Xaa(17) represents Glx; Xaa(18) represents Gly, Ala, Val,Leu, Ile, Lys, His or Arg; Xaa(19) represents Arg or Gln; Xaa(20)represents Gly, Ala, Val, Leu or Ile; Xaa(21) represents Gly, Ala, Val,Leu or Ile; Xaa(22) represents Gly, Ala, Val, Leu, Ile, Lys, H is orArg; Xaa(23) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(24)represents Gly, Ala, Val, Leu, Ile, Ser, Thr or an amino acid gap, wherein this context, an amino acid gap is understood to mean the deletion ofthat amino acid position from the polypeptide. Alternatively, amino acidreplacement at degenerate positions can be based on steric criteria,e.g. isosteric replacement, without regard for polarity or charge ofamino acid sidechains. Similarly, completely random mutagenesis of oneor more of the variant positions (Xaa) can be carried out.

In a preferred embodiment, the combinatorial CCR library is produced byway of a degenerate library of genes encoding a library of polypeptideswhich each include at least a portion of potential CCR-proteinsequences. For instance, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential CCR nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins (e.g.for phage display) containing the set of CCR protein sequences therein.

There are many ways by which the library of potential CCR homologs canbe generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then be ligated intoan appropriate gene for expression. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential CCR sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Italcura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and, forthat matter, for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of CCR homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate sequences created by combinatorialmutagenesis techniques.

In an illustrative embodiment of a screening assay, the candidatecombinatorial gene products are displayed on the surface of a cell, andthe ability of particular cells or viral particles to bind a CDK, suchas CDK4 or CDK6, via this gene product is detected in a “panning assay”.For instance, the gene library can be cloned into the gene for a surfacemembrane protein of a bacterial cell (Ladner et al., WO 88/06630; Fuchset al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS18:136-140), and the resulting fusion protein detected by panning, e.g.using a fluorescently labeled molecule which binds the CCR-protein, e.g.FITC-CDK4, to score for potentially functional CCR homologs. Cells canbe visually inspected and separated under a fluorescence microscope, or,where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In a similar fashion, the gene library can be expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, a large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and fl are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffths et al. (1993) EMBO J. 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

In an illustrative embodiment, the recombinant phage antibody system(RPAS, Pharmacia Catalog number 27-9400-01) can be easily modified foruse in expressing and screening CCR combinatorial libraries of thepresent invention. For instance, the pCANTAB 5 phagemid of the RPAS kitcontains the gene which encodes the phage gIII coat protein. The CCRcombinatorial gene library can be cloned into the phagemid adjacent tothe gill signal sequence such that it will be expressed as a gIII fusionprotein. After ligation, the phagemid is used to transform competent E.coli TG1 cells. Transformed cells are subsequently infected with M13KO7helper phage to rescue the phagemid and its candidate CCR-gene insert.The resulting recombinant phage contain phagemid DNA encoding a specificcandidate CCR-protein, and display one or more copies of thecorresponding fusion coat protein. The phage-displayed candidateproteins which are capable of, for example, binding a CDK, are selectedor enriched by panning. For instance, the phage library can be panned onglutathione immobilized CDK-GST fusion proteins, and unbound phagewashed away from the cells. The bound phage is then isolated, and if therecombinant phage express at least one copy of the wild type gill coatprotein, they will retain their ability to infect E. coli. Thus,successive rounds of reinfection of E. coli, and panning will greatlyenrich for CCR homologs, e.g. p16, p15 or p13.5 homologs, which can thenbe screened for further biological activities in order to differentiateagonists and antagonists. Subsequent selection, e.g. of a reduced set ofvariants from the library, may then be based upon more meaningfulcriteria rather than simple CDK-binding ability. For instance,intracellular half-life or inhibitory potency can become selectioncriteria in secondary screens.

In light of the present disclosure, other forms of mutagenesis generallyapplicable will be apparent to those skilled in the art in addition tothe aforementioned combinatorial mutagenesis. For example, p16, p15 orp13.5 homologs (both agonist and antagonist forms) can be generated andscreened using, for example, alanine scanning mutagenesis and the like(Ruf et al. (1994) Biochemistry 33:1565-1572; Wang et al. (1994) J.Biol. Chem. 269:3095-3099; Balint et al. (1993) Gene 137:109-118;Grodberg et al. (1993) Eur. J. Biochem. 218:597-601; Nagashima et al.(1993) J. Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry30:10832-10838; and Cunningham et al. (1989) Science 244:1081-1085), bylinker scanning mutagenesis-(Gustin et al. (1993) Virology 193:653-660;Brown et al. (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al. (1982)Science 232:316); by saturation mutagenesis (Meyers et al. (1986)Science 232:613); by PCR mutagenesis (Leung et al. (1989) Method CellMol Biol 1:11-19); or by random mutagenesis (Miller et al. (1992) AShort Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,N.Y.; and Greener et al. (1994) Strategies in Mol Biol 7:32-34).

Consequently, the invention also provides for reduction of the subjectCCR-proteins to generate mimetics, e.g. peptide or non-peptide agents,which are able to mimic binding of the authentic CCR protein to a cyclindependent kinase, e.g. CDK4 and/or CDK6. Such mutagenic techniques asdescribed above, as well as the thioredoxin system, are alsoparticularly useful for mapping the determinants of a CCR-protein whichparticipate in protein-protein interactions involved in, for example,binding of the subject CCR-protein to a CDK. To illustrate, the criticalresidues of a subject CCR-protein which are involved in molecularrecognition of CDK4 can be determined and used to generate CCR-derivedpeptidomimetics which bind to CDK4 or CDK6 and, like the authenticCCR-protein, inhibit activation of the kinase. By employing, forexample, scanning mutagenesis to map the amino acid residues of aparticular CCR-protein involved in binding a cyclin dependent kinase,peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives)can be generated which mimic those residues in binding to the kinase.For instance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in in Peptides:Structure and Function (Proceedings of the 9th American PeptideSymposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turn dipeptidecores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al.(1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols (Gordon etal. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986)Biochem Biophys Res Commun 134:71).

In a similar fashion, identification of mutations in CDK4 and/or CDK6which effect binding to a CCR-protein can be used to identify potentialpeptidyl fragments of CDK4/CDK6 which can competitively bind aCCR-protein and interfere with its ability to inhibit the CDK. Asdescribe below, we have characterized a CDK4 mutant which abrogatesbinding by p15 and p16, and consequently becomes insensitive toinhibition by those CCR-proteins. This mutation, in fact, occurs in astretch of amino acid residues which is conserved between CDK4 and CDK6.Accordingly, peptidomimetics based on this stretch might be useful asantagonists of CCR-proteins, in that they are expected to compete withCDK4 or CDK6 for binding to the CCR-protein. In a preferred embodiment,the CCR antagonist is a peptide or non-peptide analog of the amino acidsequence VAEIG(V/E)GAYG(T/K)-V(F/Y)KARD (SEQ ID NO: 15), though morepreferably a peptidomimetic of the amino acid sequence V(F/Y)KARD (SEQID NO: 16), and even more preferably of the tetrapeptide KARD (SEQ IDNO: 17). These and other peptidyl portions of CDKs can be tested forbinding to CCR-proteins such as p15 or p16 using, for example, thethioredoxin fusion proteins constructs mention above.

Another aspect of the invention pertains to an antibody specificallyreactive with one of the subject CCR-proteins. For example, by usingpeptides based on the cDNA sequence of the subject p16 protein, anti-p16antisera or anti-p16 monoclonal antibodies can be made using standardmethods. Likewise, anti-p13.5 and anti-p15 antibodies can be generated.A mammal such as a mouse, a hamster or rabbit can be immunized with animmunogenic form of the peptide (e.g., an antigenic fragment which iscapable of eliciting an antibody response). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. For instance, a peptidylportion of the protein represented by one of SEQ ID NO: 2, 4 or 6 can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization, anti-CCR antisera can be obtained and, ifdesired, polyclonal anti-CCR antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), as the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with the CCR-protein ofinterest and the monoclonal antibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with a CCR-protein, e.g.anti-p16, anti-p5 or anti-p13.5 antibodies. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described above for whole antibodies. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules.

Both monoclonal and polyclonal antibodies (Ab) directed against thesubject CCR-proteins, and antibody fragments such as Fab′ and F(ab′)₂,can be used to block the action of particular CCR and allow the study ofthe cell-cycle or cell proliferation.

One application of anti-CCR antibodies is in the immunological screeningof cDNA libraries constructed in expression vectors, such as λgt11,λgt18-23, λZAP, and λORF8. Messenger libraries of this type, havingcoding sequences inserted in the correct reading frame and orientation,can produce fusion proteins. For instance, λgt11 will produce fusionproteins whose amino termini consist of β-galactosidase amino acidsequences and whose carboxy termini consist of a foreign polypeptide.Antigenic epitopes of a CCR-protein, such as proteins antigenicallyrelated to p16, p15 or p13.5, can then be detected with antibodies, as,for example, reacting nitrocellulose filters lifted from infected plateswith an anti-CCR antibody. Phage, scored by this assay, can then beisolated from the infected plate. Thus, the presence of CCR homologs,such as p16, p15 or p13.5 homologs, can be detected and cloned fromother sources.

Antibodies which are specifically immunoreactive with one or moreCCR-proteins of the present invention can also be used inimmunohistochemical staining of tissue samples in order to evaluate theabundance and pattern of expression of the CCR-protein family, orparticular members thereof. Anti-CCR antibodies can be useddiagnostically in immuno-precipitation and immuno-blotting to detect andevaluate levels of one or more CCR-proteins in tissue or cells isolatedfrom a bodily fluid as part of a clinical testing procedure. Forinstance, such measurements can be useful in predictive valuations ofthe onset or progression of tumors. Likewise, the ability to monitorcertain CCR-protein levels in an individual can allow determination ofthe efficacy of a given treatment regimen for an individual afflictedwith such a disorder. Diagnostic assays using anti-CCR antibodies, suchas anti-p16 or anti-p15 antibodies, can include, for example,immunoassays designed to aid in early diagnosis of a neoplastic orhyperplastic disorder, e.g. the presence of cancerous cells in thesample, e.g. to detect cells in which a lesion of a CCR-gene hasoccurred.

In addition, nucleotide probes can be generated from the cloned sequenceof the subject CCR-proteins, which allow for histological screening ofintact tissue and tissue samples for the presence of a CCR-proteinencoding mRNA. Similar to the diagnostic uses of anti-CCR-proteinantibodies, the use of probes directed to CCR-protein encoding mRNAs, orto genomic CCR-gene sequences, can be used for both predictive andtherapeutic evaluation of allelic mutations which might be manifest in,for example, neoplastic or hyperplastic disorders (e.g. unwanted cellgrowth). Used in conjunction with anti-CCR protein antibodyimmunoassays, the nucleotide probes can help facilitate thedetermination of the molecular basis for a developmental disorder whichmay involve some abnormality associated with expression (or lackthereof) of a CCR-protein. For instance, variation in CCR-proteinsynthesis can be differentiated from a mutation in the coding sequence.

Accordingly, the present method provides a method for determining if asubject is at risk for a disorder characterized by unwanted cellproliferation. In preferred embodiments, method can be generallycharacterized as comprising detecting, in a tissue of said subject, thepresence or absence of a genetic lesion characterized by at least one of(i) a mutation of a gene encoding a CCR-protein, such as p16, p15 orp13.5 or (ii) the mis-expression of the CCR-gene. To illustrate, suchgenetic lesions can be detected by ascertaining the existence of atleast one of (i) a deletion of one or more nucleotides from a CCR-gene,(ii) an addition of one or more nucleotides to a CCR-gene, (iii) asubstitution of one or more nucleotides of a CCR-gene, (iv) a grosschromosomal rearrangement of a CCR-gene, (v) a gross alteration in thelevel of a messenger RNA transcript of a CCR-gene, (vi) the presence ofa non-wild type splicing pattern of a messenger RNA transcript of aCCR-gene, and (vii) a non-wild type level of a CCR-protein. In oneaspect of the invention, there is provided a probe/primer comprising anoligonucleotide containing a region of nucleotide sequence which iscapable of hybridizing to a sense or antisense sequence of any of SEQ IDNOs: 1, 3 or 5 or naturally occurring mutants thereof, or 5′ or 3′flanking sequences or intronic sequences naturally associated with thesubject CCR-genes or naturally occurring mutants thereof. The probe isexposed to nucleic acid of a tissue sample; and the hybridization of theprobe to the sample nucleic acid is detected. In certain embodiments,detection of the lesion comprises utilizing the probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1944) PNAS 91:360-364), the later of which can be particularly usefulfor detecting point mutations in the CCR-gene. Alternatively, the levelof CCR-protein can be detected in an immunoassay.

Moreover, the use of anti-sense techniques (e.g. microinjection ofantisense molecules, or transfection with plasmids whose transcripts areanti-sense with regard to a certain CCR mRNA) can be used to investigaterole of particular CCR-proteins, e.g. p16, p15 or p13.5, in thecell-cycle and cell proliferation, in a controlled environment, byinhibiting endogenous production of the protein. Such techniques can beutilized in cell culture, but can also be used in the creation oftransgenic animals.

Another aspect of the invention features transgenic non-human animalswhich express a heterologous CCR-gene of the present invention, or whichhave had one or more genomic CCR-gene(s), disrupted in at least one ofthe tissue or cell-types of the animal. For instance, transgenic micewhich have been disrupted at their p13.5 gene locus are described inExample 5.

In another aspect, the invention features an animal model fordevelopmental diseases, which has a CCR allele which is mis-expressed.For example, a mouse can be bred which has a p16 or p15 allele deleted,or in which all or part of one or more p16 exons are deleted. Such amouse model can then be used to study disorders arising frommis-expressed p16 genes.

Another aspect of the present invention concerns transgenic animalswhich are comprised of cells (of that animal) which contain a transgeneof the present invention and which preferably (though optionally)express an exogenous CCR-protein in one or more cells in the animal. TheCCR-transgene can encode the wild-type form of the protein, or canencode homologs thereof, including both agonists and antagonists, aswell as antisense constructs. In preferred embodiments, the expressionof the transgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosaic expression of the subject proteins can be essential for manyforms of lineage analysis and can additionally provide a means to assessthe effects of, for example, lack of p16 and p15 inhibition of CDKswhich might grossly alter development in small patches of tissue withinan otherwise normal embryo. Toward this and, tissue-specific regulatorysequences and conditional regulatory sequences can be used to controlexpression of the transgene in certain spatial patterns. Moreover,temporal patterns of expression can be provided by, for example,conditional recombination systems or prokaryotic transcriptionalregulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of the subject CCR polypeptide. For example, excision of atarget sequence which interferes with the expression of a recombinantCCR-gene can be designed to activate expression of that gene. Thisinterference with expression of the protein can result from a variety ofmechanisms, such as spatial separation of the CCR-gene from the promoterelement or an internal stop codon. Moreover, the transgene can be madewherein the coding sequence of the gene is flanked recombinancerecognition sequences and is initially transfected into cells in a 3′ to5′ orientation with respect to the promoter element. In such aninstance, inversion of the target sequence will reorient the subjectgene by placing the 5′ end of the coding sequence in an orientation withrespect to the promoter element which allow for promoter driventranscriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotiderepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of the CCR protein can be regulated viaregulation of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant CCR protein, such as p15 or p16, requires the constructionof a transgenic animal containing transgenes encoding both the Crerecombinase and the subject protein. Animals containing both the Crerecombinase and the recombinant CCR genes can be provided through theconstruction of “double” transgenic animals. A convenient method forproviding such animals is to mate two transgenic animals each containinga transgene, e.g., the CCR-gene and recombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a CCR-transgene in a recombinase-mediated expressible formatderives from the likelihood that the subject protein will be deleteriousupon expression in the transgenic animal. In such an instance, a founderpopulation, in which the subject transgene is silent in all tissues, canbe propagated and maintained. Individuals of this founder population canbe crossed with animals expressing the recombinase in, for example, oneor more tissues. Thus, the creation of a founder population in which,for example, an antagonistic p15 transgene is silent will allow thestudy of progeny from that founder in which disruption of cell cycleregulation by p15 in a particular tissue or at developmental stageswould result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080. Moreover, expression of theconditional transgenes can be induced by gene therapy-like methodswherein a gene encoding the trans-activating protein, e.g. a recombinaseor a prokaryotic protein, is delivered to the tissue and caused to beexpressed, such as in a cell-type specific manner. By this method, theCCR-transgene could remain silent into adulthood until “turned on” bythe introduction of the trans-activator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic non-human animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Microinjection ofzygotes is the preferred method for incorporating transgenes inpracticing the invention.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

Methods of making knock-out or disruption transgenic animals are alsogenerally known. See, for example, Manipulating the Mouse Embryo, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a CCR-gene can becontrolled as above.

Yet another aspect of the invention pertains to methods of treatingproliferative and/or differentiative disorders which arise from cellswhich, despite aberrant growth control, still require CDK4 or CDK6 forcell growth. There are a wide variety of pathological cell proliferativeconditions for which the CCR-gene constructs and CCR-mimetics of thepresent invention can provide therapeutic benefits, with the generalstrategy being the inhibition of an anomalous cell proliferation. Forinstance, the gene constructs of the present invention can be used as apart of a gene therapy protocol, such as to reconstitute the function ofa CCR-protein, e.g. p16, p15 or p13.5, in a cell in which the protein ismisexpressed or in which signal transduction pathways upstream of theCCR-protein are dysfunctional. To illustrate, cell types which exhibitpathological or abnormal growth presumably dependent at least in part ona function of a CCR-protein include various cancers and leukemias,psoriasis, bone diseases, fibroproliferative disorders such as involvingconnective tissues, atherosclerosis and other smooth muscleproliferative disorders, as well as chronic inflammation. In addition toproliferative disorders, the treatment of differentiative disorderswhich result from, for example, de-differentiation of tissue which may(optionally) be accompanied by abortive reeentry into mitosis. Suchdegenerative disorders include chronic neurodegenerative diseases of thenervous system, including Alzheimer's disease, Parkinson's disease,Huntington's chorea, amylotrophic lateral sclerosis and the like, aswell as spinocerebellar degenerations. Other differentiative disordersinclude, for example, disorders associated with connective tissue, suchas may occur due to de-differentiation of chondrocytes or osteocytes, aswell as vascular disorders which involve de-differentiation ofendothelial tissue and smooth muscle cells, gastric ulcers characterizedby degenerative changes in glandular cells, and renal conditions markedby failure to differentiate, e.g. Wilm's tumors. It will also beapparent that, by transient use of gene therapy constructs of thesubject CCR proteins (e.g. agonist and antagonist forms), in vivoreformation of tissue can be accomplished, e.g. in the development andmaintenance of organs. By controlling the proliferative anddifferentiative potential for different cells, the subject geneconstructs can be used to reform injured tissue, or to improve graftingand morphology of transplanted tissue. For instance, CCR agonists andantagonists can be employed therapeutically to regulate organsafter-physical, chemical or pathological insult. For example, genetherapy can be utilized in liver repair subsequent to a partialhepatectomy, or to promote regeneration of lung tissue in the treatmentof emphysema.

For instance, as described in the Examples below, transformation of acell can be due in part to a loss-of-function mutation to a particularCCR-gene, e.g., ranging from a point mutation to gross deletion of thegene. Additionally, other data provided in the appended examplessuggests that disorders susceptible to treatment with CCR agonistsinclude those arising from cells which have lost the ability to induceCCR-protein expression. Normal cell proliferation, for instance, isgenerally marked by responsiveness to negative autocrine or paracrinegrowth regulators, such as members of the TGF-β family, e.g. TGF-β1,TGF-β2 or TGF-β3, and related polypeptide growth inhibitors, e.g.activins, inhibins, Mullerian inhibiting substance, decapentaplegic,bone morphogenic factors, and vg1 (e.g. terminal differentiationinducers). Ordinarily, control of cellular proliferation by such growthregulators, particularly in epithelial and hemopoietic cells, is in theform of growth inhibition. This is generally accompanied bydifferentiation of the cell to a post-mitotic phenotype. However, it hasbeen observed that a significant percentage of human cancers derivedfrom these cells types display a reduced responsiveness to growthregulators such as TGF-β. For instance, some tumors of colorectal, liverepithelial, and epidermal origin show reduced sensitivity and resistanceto the growth-inhibitory effects of TGF-β as compared to their normalcounterparts. In this context, a noteworthy characteristic of severalretinoblastoma cell lines is the absence of detectable TGF-β receptors.Treatment of such tumors with CCR agonists (e.g. CCR-proteins deliveredby gene therapy or CCR-mimetics) provides an opportunity to restore thefunction of the RB-mediated checkpoint.

However, it will be appreciated that the subject method can be used toinhibit proliferation of cells which, in general, are still reliant oncyclin dependent kinases active in G₁, e.g. CDK4 or CDK6, irrespectiveof involvement of RB or RB-like proteins.

In accordance with the subject method, expression constructs of thesubject CCR-proteins may be administered in any biologically effectivecarrier, e.g. any formulation or composition capable of effectivelytransfecting cells in vivo with a recombinant CCR-gene. Approachesinclude insertion of the subject gene in viral vectors includingrecombinant retroviruses, adenovirus, adeno-associated virus, and herpessimplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viralvectors can be used to transfect cells directly; plasmid DNA can bedelivered with the help of, for example, cationic liposomes (lipofectin)or derivatized (e.g. antibody conjugated), polylysine conjugates,gramicidin S, artificial viral envelopes or other such intracellularcarriers, as well as direct injection of the gene construct or CaPO₄precipitation carried out in vivo. It will be appreciated that becausetransduction of appropriate target cells represents the critical firststep in gene therapy, choice of the particular gene delivery system willdepend on such factors as the phenotype of the intended target and theroute of administration, e.g. locally or systemically.

A preferred approach for in vivo introduction of nucleic acid encodingone of the subject proteins into a cell is by use of a viral vectorcontaining nucleic acid, e.g. a cDNA, encoding the gene product.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding oneof the subject CCR-proteins, rendering the retrovirus replicationdefective. The replication defective retrovirus is then packaged intovirions which can be used to infect a target cell through the use of ahelper virus by standard techniques. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14 and other standard laboratory manuals. Examples of suitableretroviruses include pLJ, pZIP, pWE and pEM which are well known tothose skilled in the art. Examples of suitable packaging virus lines forpreparing both ecotropic and amphotropic retroviral systems include.psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used tointroduce a variety of genes into many different cell types, includingneural cells, epithelial cells, endothelial cells, lymphocytes,myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (seefor example Eglitis, et al. (1985) Science 230:1395-1398; Danos andMulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990)Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl.Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci.USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (993) J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; andPCT Application WO 92/07573).

In choosing retroviral vectors as a gene delivery system for the subjectCCR-genes, it is important to note that a prerequisite for thesuccessful infection of target cells by most retroviruses, and thereforeof stable introduction of the recombinant CCR-gene, is that the targetcells must be dividing. In general, this requirement will not be ahindrance to use of retroviral vectors to deliver CCR-gene constructs.In fact, such limitation on infection can be beneficial in circumstanceswherein the tissue (e.g. nontransformed cells) surrounding the targetcells does not undergo extensive cell division and is thereforerefractory to infection with retroviral vectors.

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al. (1992) J.Gen Virol 73:3251-3255; and Goud et al. (1983) Virology 163:251-254); orcoupling cell surface ligands to the viral env proteins (Neda et al.(1991) J Biol Chem 266:14143-14146). Coupling can be in the form of thechemical cross-linking with a protein or other variety (e.g. lactose toconvert the env protein to an asialoglycoprotein), as well as bygenerating fusion proteins (e.g. single-chain antibody/env fusionproteins). This technique, while useful to limit or otherwise direct theinfection to certain tissue types, and can also be used to convert anecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the CCR-gene of the retroviral vector.

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes a gene product of interest, but isinactivate in terms of its ability to replicate in a normal lytic virallife cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616;Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992)Cell 68:143-155). Suitable adenoviral vectors derived from theadenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.Recombinant adenoviruses can be advantageous in certain circumstances inthat they are not capable of infecting nondividing cells and can be usedto infect a wide variety of cell types, including airway epithelium(Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand etal. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486); hepatocytes (Herzand Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and musclecells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al., supra; Haj-Ahmand andGraham (1986) J. Virol. 57:267). Most replication-defective adenoviralvectors currently in use and therefore favored by the present inventionare deleted for all or parts of the viral E1 and E3 genes but retain asmuch as 80% of the adenoviral genetic material (see, e.g., Jones et al.(1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methodsin Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991)vol. 7. pp. 109-127). Expression of the inserted CCR-gene can be undercontrol of, for example, the E1A promoter, the major late promoter (MLP)and associated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

Yet another viral vector system useful for delivery of the subjectCCR-gene is the adeno-associated virus (AAV). Adeno-associated virus isa naturally occurring defective virus that requires another virus, suchas an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulsld et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470.Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

Other viral vector systems that may have application in gene therapyhave been derived from herpes virus, vaccinia virus, and several RNAviruses. In particular, herpes virus vectors may provide a uniquestrategy for persistence of the recombinant CCR-gene in cells of thecentral nervous system and ocular tissue (Pepose et al. (1994) InvestOpthalmol Vis Sci 35:2662-2666)

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of aCCR-protein in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject CCR-gene by thetargeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

In a representative embodiment, a gene encoding one of the subjectCCR-proteins can be entrapped in liposomes bearing positive charges ontheir surface (e.g., lipofectins) and (optionally) which are tagged withantibodies against cell surface antigens of the target tissue (Mizuno etal. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309;Japanese patent application 1047381; and European patent publicationEP-A-43075). For example, lipofection of neuroglioma cells can becarried out using liposomes tagged with monoclonal antibodies againstglioma-associated antigen (Mizuno et al. (1992) Neurol. Med. Chir.32:873-876).

In yet another illustrative embodiment, the gene delivery systemcomprises an antibody or cell surface ligand which is cross-linked witha gene binding agent such as polylysine (see, for example, PCTpublications WO93/04701, WO92/22635, WO92/20316, WO92/19749, andWO92/06180). For example, the subject CCR-gene construct can be used totransfect hepatocytic cells in vivo using a soluble polynucleotidecarrier comprising an asialoglycoprotein conjugated to a polycation,e.g. poly-lysine (see U.S. Pat. No. 5,166,320). It will also beappreciated that effective delivery of the subject nucleic acidconstructs via mediated endocytosis can be improved using agents whichenhance escape of the gene from the endosomal structures. For instance,whole adenovirus or fusogenic peptides of the influenza HA gene productcan be used as part of the delivery system to induce efficientdisruption of DNA-containing endosomes (Mulligan et al. (1993) Science260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano et al. (1993)PNAS 90:2122).

In clinical settings, the gene delivery systems can be introduced into apatient by any of a number of methods, each of which is familiar in theart. For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g. by intravenous injection,and specific transduction of the in the target cells occurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of the gene,or a combination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (e.g. Chen et al. (1994) PNAS 3054-3057).

Moreover, the pharmaceutical preparation can consist essentially of thegene delivery system in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery system can be producedin tact from recombinant cells, e.g. retroviral packages, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system. In the case of the latter, methods ofintroducing the viral packaging cells may be provided by, for example,rechargeable or biodegradable devices. Various slow release polymericdevices have been developed and tested in vivo in recent years for thecontrolled delivery of drugs, including proteinaceousbiopharmaceuticals, and can be adapted for release of viral particlesthrough the manipulation of the polymer composition and form. A varietyof biocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of an the viral particles by cellsimplanted at a particular target site. Such embodiments of the presentinvention can be used for the delivery of an exogenously purified virus,which has been incorporated in the polymeric device, or for the deliveryof viral particles produced by a cell encapsulated in the polymericdevice.

By choice of monomer composition or polymerization technique, the amountof water, porosity and consequent permeability characteristics can becontrolled. The selection of the shape, size, polymer, and method forimplantation can be determined on an individual basis according to thedisorder to be treated and the individual patient response. Thegeneration of such implants is generally known in the art. See, forexample, Concise Encyclopedia of Medical & Dental Materials, ed. byDavid Williams (MIT Press: Cambridge, Mass., 1990); and the Sabel et al.U.S. Pat. No. 4,883,666. In another embodiment of an implant, a sourceof cells producing a the recombinant virus is encapsulated inimplantable hollow fibers. Such fibers can be pre-spun and subsequentlyloaded with the viral source (Aebischer et al. U.S. Pat. No. 4,892,538;Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al. (1990) Expt.Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46;and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or can beco-extruded with a polymer which acts to form a polymeric coat about theviral packaging cells (Lim U.S. Pat. No. 4,391,909; Sefton U.S. Pat. No.4,353,888; Sugamori et al. (1989) Trans. Am. Artif Intern. Organs35:791-799; Sefton et al. (1987) Biotechnol. Bioeng. 29:1135-1143; andAebischer et al. (1991) Biomaterials 12:50-55). Again, manipulation ofthe polymer can be carried out to provide for optimal release of viralparticles.

To further illustrate the use of the subject method, the therapeuticapplication of a CCR-gene, e.g., by gene therapy, can be used in thetreatment of a neuroglioma. Gliomas account for 40-50% of intracranialtumors at all ages of life. Despite the increasing use of radiotherapy,chemotherapy, and sometimes immunotherapy after surgery for malignantglioma, the mortality and morbidity rates have not substantiallyimproved. However, there is increasing experimental and clinicalevidence that for a significant number of gliomas, loss of TGF-βresponsiveness is an important event in the loss of growth control.Irrespective of the cause of decreased responsiveness, e.g. the loss offunction of p15 or the loss of other TGF-β signal transduction proteins,exogenous expression of p15 (or other CCR-protein) in the cell can usedeffectively to inhibit cell proliferation.

It has been demonstrated that gene therapy can be used to target gliomacells for expression of recombinant proteins (Miyao et al. (1993) J.Neurosci. Res. 36:472-479; Chen et al. (1994) PNAS 91:3054-3057; andTakamiya et al. (1993) J. Neurosurg 79:104-110). Thus, a gene constructfor expressing a CCR-protein can be delivered to the tumor, preferablyby stereotactic-dependent means. In preferred embodiments, the genedelivery system is a retroviral vector. Since rapidly growing normalcells are rare in the adult CNS, glioma cells can be specificallytransduced with a recombinant retrovirus. For example, the retroviralparticle can be delivered into the tumor cavity through an Ommaya tubeafter surgery, or alternatively, packaging fibroblasts encapsulated inretrievable immunoisolatory vehicles can be introduced into the tumorcavity. In order to increase the effectiveness and decrease the sideeffects of the retrovirus-mediated gene therapy, glioma-specificpromoters can be used to regulate expression of the CCR-gene. Forexample, the promoter regions of glial fibrillary acidic protein (GFAP)and myelin basis protein (MBP) can operably linked to the recombinantgene in order to direct glial cell-specific expression of therecombinant gene construct.

In another embodiment, gene therapy can be used in conjunction with thesubject CCR-protein in the treatment of various carcinomas. Among thecarcinomas, a strong correlation has been observed between TGF-βresponsiveness and both increasing malignant tumor grade and increasingproliferating cell nuclear antigen index (Coffery et al. (1988) CancerRes 48:1596-1602; Masui et al. (1986) PNAS 83:2438-2442; and Knabble etal. (1987) Cell 48:417-428). In a representative embodiment, a genetherapy system comprising the subject CCR-gene in a retroviral vector isused to treat certain breast cancers. In preferred embodiments,expression of the subject CCR-protein-gene is controlled at least inpart by a mammary-specific promoter, a number of which are available(for review, see Hennighausen (1990) Protein Expression and Purification1:3-8; and Gunzberg et al. (1992) Biochem J 283:625-632).

In similar fashion, gene therapy protocols involving delivery of thesubject CCR-gene can be used in the treatment of malignant melanoma,which also serves as a model for progressive TGF-β resistance intransformation. In preferred embodiments, gene therapy protocols fortreatment of melanomas include, in addition to the delivery of theCCR-gene construct by retroviral delivery, the delivery of apharmaceutical preparation of the gene by direct injection. Forinstance, U.S. Pat. No. 5,318,514 describes an applicator for theelectroporation of genes into epidermal cells and can be used inaccordance with the present invention.

The subject CCR-gene constructs can be used in the treatment ofhyperproliferative vascular disorders, e.g. smooth muscle hyperplasia(such as atherosclerosis) or restinosis, as well as other disorderscharacterized by fibrosis, e.g. rheumatoid arthritis, insulin dependentdiabetes mellitus, glomerulonephritis, cirrhosis, and scleroderma,particularly proliferative disorders in which loss of TGF-β autocrine orparacrine signalling is implicated.

For example, restinosis continues to limit the efficacy of coronaryangioplasty despite various mechanical and pharmaceutical interventionsthat have been employed. An important mechanism involved in normalcontrol of intimal proliferation of smooth muscle cells appears to bethe induction of autocrine and paracrine TGF-β inhibitory loops in thesmooth muscle cells (Scott-Burden et al. (1994) Tex Heart Inst J21:91-97; Graiger et al. (1993) Cardiovasc Res 27:2238-2247; andGrainger et al. (1993) Biochem J 294:109-112). Loss of sensitivity toTGF-β, or alternatively, the overriding of this inhibitory stimulus suchas by PDGF autostimulation, can be a contributory factor to abnormalsmooth muscle proliferation in restinosis. It may therefore be possibleto treat or prevent restinosis by the use of gene therapy with CCR-geneconstructs of the present invention. The CCR-gene construct can bedelivered, for example, by percutaneous transluminal gene transfer(Mazur et al. (1994) Tex Heart Inst J 21:104-111) using viral orliposomal delivery compositions. An exemplary adenovirus-mediated genetransfer technique and compositions for treatment of cardiac or vascularsmooth muscle is provided in PCT publication WO 94/11506.

Transforming growth factors β is also understood to play a significantrole in local glomerular and interstitial sites in human kidneydevelopment and disease. Consequently, the subject method provides amethod of treating or inhibiting glomerulopathies and other renalproliferative disorders comprising the in vivo delivery and recombinantexpression of the subject CCR-protein in kidney tissue.

The subject method can also be used to treat retinoblastomas in whichthe retinoblastoma gene (RB) is not itself impaired, e.g. the effectiveimpairment of the RB checkpoint is the result of a failure to controlCDK4 phosphorylation of RB. Thus, an exogenous CCR-gene can be expressedin a retinoblastoma cell, thereby causing inhibition of CDK4 activationand down-regulating RB phosphorylation. To illustrate, a recombinantretrovirus can be constructed to facilitate expression of p16 or p15,and infectivity of retinoblastoma cells enhanced by derivatizing the envprotein with antibodies specific for retinoblastoma cells, e.g.antibodies to retinal S-antigen (Doroso et al. (1985) Invest OpthalmolVis Sci 26:560-572; see also Liao et al. (1981) Eur J Immunol11:450-454; and U.S. Pat. No. 4,444,744).

In yet another embodiment, the subject CCR-gene is delivered to asarcoma, e.g. an osteosarcoma or Kaposi's sarcoma. In a representativeembodiment, the gene is provided in a viral vector and delivered by wayof a viral particle which has been derivatized with antibodiesimmunoselective for an osteosarcoma cell (see, for example, U.S. Pat.Nos. 4,564,517 and 4,444,744; and Singh et al. (1976) Cancer Res36:4130-4136).

In a still further embodiment, the subject CCR-gene is recombinantlyexpressed in tissue which is characterized by unwantedde-differentiation and which may also be undergoing unwanted apoptosis.For instance, many neurological disorders are associated withdegeneration of discrete populations of neuronal elements. For example,Alzheimer's disease is associated with deficits in severalneurotransmitter systems, both those that project to the neocortex andthose that reside with the cortex. For instance, the nucleus bacillus inpatients with Alzheimer's disease were observed to have a profound (75%)loss of neurons compared to age-matched controls. Although Alzheimer'sdisease is by far the most common form of dementia, several otherdisorders can produce dementia. Many are age-related, occurring in fargreater incidence in older people than in younger. Several of these aredegenerative diseases characterized by the death of neurons in variousparts of the central nervous system, especially the cerebral cortex.However, some forms of dementia are associated with degeneration of thethalamus or the white matter underlying the cerebral cortex. Here, thecognitive dysfunction results from the isolation of cortical areas bythe degeneration of efferents and afferents. Huntington's diseaseinvolves the degeneration of intrastraital and cortical cholinergicneurons and GABAergic neurons. Pick's disease is a severe neuronaldegeneration in the neocortex of the frontal and anterior temporallobes, sometimes accompanied by death of neurons in the striatum.Accordingly, the subject CCR-gene can be delivered to the effectedtissue by gene therapy techniques. It is noted that numerous advanceshave been made in the construction of expression vectors, cellular andviral transgene carriers, and the characterization of target cells forneuronal gene therapy, and can be readily adapted for delivery of thesubject CCR-genes (see, for example, Suhr et al. (1993) Arch Neurol50:1252-1268; Jiao et al. (1993) Nature 362:450-453; Friedmann (1992)Ann Med 24:411-417; and Freese et al. (1991) Nuc Acid Res 19:7219-7223)

In addition to degenerative-induced dementias, the subject gene therapysystems can be applied opportunely in the treatment of neurodegenerativedisorders which have manifestations of tremors and involuntarymovements. Parkinson's disease, for example, primarily affectssubcortical structures and is characterized by degeneration of thenigrostriatal pathway, raphe nuclei, locus cereleus, and the motornucleus of vagus. Ballism is typically associated with damage to thesubthalamic nucleus, often due to acute vascular accident. Also includedare neurogenic and myopathic diseases which ultimately affect thesomatic division of the peripheral nervous system and are manifest asneuromuscular disorders. Examples include chronic atrophies such asamyotrophic lateral sclerosis, Guillain-Barre syndrome and chronicperipheral neuropathy, as well as other diseases which can be manifestas progressive bulbar palsies or spinal muscular atrophies. Moreover,the use of CCR gene therapy constructs is amenable to the treatment ofdisorders of the cerebellum which result in hypotonia or ataxia, such asthose lesions in the cerebellum which produce disorders in the limbsipsilateral to the lesion. For instance, p16 or p15 gene constructs canused to treat a restricted form of cerebellar corical degenerationinvolving the anterior lobes (vermis and leg areas) such as is common inalcoholic patients.

Furthermore, the subject CCR-genes can also be used in the treatment ofautonomic disorders of the peripheral nervous system, which includedisorders affecting the innervation of smooth muscle and endocrinetissue (such as glandular tissue). For instance, recombinant p16 or p15expression by gene therapy can be used to treat tachycardia or atrialcardiac arrythmias which may arise from a degenerative condition of thenerves innervating the striated muscle of the heart.

Yet another aspect of the invention pertains to CDK4 and CDK6 mutantswhich are insensitive to the inhibitory control of one or more of thesubject CCR proteins. With respect to cell cycle control, these mutantsantagonize the action of CCR-proteins, and in preferred embodiments, aredominant negative mutants in that they remove from the control ofmitosis certain cell-cycle checkpoints, such as checkpoints mediated byRB or RB-like proteins. Exemplary CDK4 and CDK6 mutants are representedin SEQ ID NOs: 9 (CDK4) and SEQ ID NOs: 10 (CDK6), wherein Xaa, which isan Arg in the wild-type enzyme, is mutated to render the enzymeinsensitive to inhibition by CCR proteins. In preferred embodiments, Xaais an amino acid other than arginine, though more preferably an aminoacid which does not have a basic sidechain (e.g. not Arg, Lys or His).In a preferred embodiment, the Arginine is changed to a Cysteine.

The mutant CDK4 and CDK6 proteins, genes encoding such proteins, andantibodies specific for such proteins, can be provided in a manneranalogous to any of the embodiments described above for each of theCCR-proteins. For instance, nucleic acids encoding the mutant kinasescan be provided in any of a number of forms, e.g. as recombinantexpression vectors as well as probes. Both isolated and recombinantforms of the enzymes are specifically contemplated, as well asantibodies which specifically bind the mutant forms of each of thekinases. Moreover, it will be appreciated that, wherever the presentapplication mentions the utility for CCR antagonists, the subject CDKmutants can be utilized.

While the Arg.fwdarw.Cys mutant of CDK4 was first discovered in cellsfrom a melanoma patient (see Example 10), mutagenic techniques such asthose described above can be utilized to isolate yet other CDK4 and CDK6mutants which are resistant to CCR protein inhibition. For instance,scanning mutagenesis or random mutagenesis can be used to generate alibrary of CDK mutants which can be screened for dominant negativemutations employing assays sensitive to CDK4 or CDK6 inhibition. Forexample, a library of CDK4 mutants can be transfected into cells whichoverexpress p16 and which are quiescent as a result. In the absence ofmutations which uncouple the CDK4 mutant from p16/p15, the cell willremain quiescent. In contrast, mutations to the kinase which uncoupleits activity from p16 will result in proliferation of cells expressingthat mutant of CDK4. In a similar embodiment, the level ofphosphorylated RB can be used to detect dominant negative mutants ofCDK4, such as by immunodetection of phosphorylated RB, or the use ofRB-sensitive reproter constructs the expression of which is dependent onthe phosphorylation state of RB (e.g., see Park et al. (1994) J BiolChem 269:6083-6088; Ouellette et al. (1992) Oncogene 7:1075-1081; andSlack et al. (1993) Oncogene 8:1585-1591). Mutants which uncouplep16/p15 inhibition of CDK4 will cause higher levels of phosphorylated RBprotein to accumulate in the cell.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

As described previously (see U.S. patent application Ser. Nos.08/154,915, 07/991,997 and 07/963,308, as well as Xiong et al. (1993)Nature 366:701; Xiong et al. (1993) Genes Dev 7:1572; Xiong et al.(1992) Cell 71:505; and Zhang et al. (1993) Mol Cell Biol 4:897),immunological procedures have been used to establish that cyclinsassociate, in eukaryotic cells, with a variety of potential catalyticsubunits (e.g., CDKs, such as CDK2, CDK4 and CDK5). To illustrate, humancyclin D1 has been associated with a wide variety of proliferativediseases. In human diploid cells, specifically human diploidfibroblasts, cyclin D1 is complexed with a number of other cellularproteins. Among them are the catalytic subunits CDK2, CDK4 (previouslycalled PSK-J3), CDK5 (also called PSSALRE), and CDK6 (PLSTIRE). Inaddition, polypeptides of 21 kDa and 36 kDa were identified inassociation with cyclin D1. It was shown that the 36 kDa protein is theProliferating Cell Nuclear Antigen, PCNA. PCNA has been described as anessential accessory factor to the delta polymerase, which is requiredfor leading-strand DNA replication and DNA repair. Cyclin D3 was alsofound to associate with multiple protein kinases, p21 and PCNA. It wastherefore proposed that there exists a quaternary complex of D typecyclins, CDK, PCNA and p21, and that many combinatorial variations(cyclin D1, D3 with CDK2, 4, 5 and 6) may assemble in vivo.

The importance of the quaternary complex is emphasized by the discoverythat cellular transformation by DNA tumor viruses is associated withselective subunit rearrangement of the cyclin D complexes, as well asother cell-cycle complexes, including cyclin A, CDC2, CDK2, CDK4 andCDK5 complexes. In particular, introduction of SV40 DNA tumor virus orits oncogenic gene product large T antigen into normal human diploidfibroblasts (HDF) causes disruption of the association between cyclin D-and PCNA, CDKs (such as CDK2, CDK4, CDK5 and CDK6) and p21. For example,after dissociation from cyclin D and p21, CDK4 kinase becomes associatedwith a 16 kDa polypeptide (p16). Similarly, SV40 transformation causes adecrease of association of p21 with cyclin A in HDF; and adenovirus-(293cell line) or human papilloma virus-(HeLa cell line) transformed cells,p21 is completely disassociated from cyclin A. A 19 kDa peptide, p19,then appears in a complex with cyclin A.

Thus, in many transformed cells, cyclin and CDK's associate in binarycomplexes which form the core of the cell-cycle regulatory machinery. Innormal cells, a major fraction of the cyclin kinases acquire twoadditional subunits (p21 and PCNA) and thereby form quaternarycomplexes. Reconstitution of quaternary complexes in insect cellsrevealed that p21 is a universal inhibitor of cyclin kinases. As such,p21 inhibits cell-cycle progression and cell proliferation uponoverexpression in mammalian cells. Taken in conjunction with thepreviously demonstrated absence of p21 protein in the cell-cycle kinasecomplexes of cells with deficient p53, these results suggest that p21 isa transcriptional target of the tumor suppressor protein, p53. Onefunction of p53 is to act in a cellular signaling pathway which causescell-cycle arrest following DNA damage (see for example, Kastan et al.Cell 71:587-5971993). It has therefore been suggested that p21 forms acritical link between p53 and the cell-cycle control machinery.

Cyclin D/CDK4 kinase differs from the others in its inability to utilizehistone H1 as a substrate. To date, the only substrates known for cyclinD/CDK4 kinases are the members of the RB family of “pocket” proteins(Matsushime et al., Cell 71:323-334 (1992)). Therefore, the effect ofp21 was tested on the ability of cyclin D/CDK4 to phosphorylated RB.Insect cell lysates containing cyclin D or CDK4 alone showed littleactivity toward GST-RB. However, cyclin D/CDK4 binary complexescatalyzed substantial RB phosphorylation. Addition of increasing amountsof p21 resulted in the accumulation of cyclin D/CDK4/p21 ternarycomplexes with a corresponding inhibition of RB phosphorylation.Inclusion of PCNA was essentially without effect. However, cells lackingfunctional p53 may nevertheless retain a functional RB checkpoint whichundergoes differential phosphorylation despite lack of endogenous p21.

The two-hybrid screening system (Fields et al. Nature 340:254 (1989))was utilized to search for proteins that could interact with human CDK4,and more specifically, to isolate a cDNA encoding p16. Two-hybridscreening relies on reconstituting a functional GAL4 activator from twoseparated fusion proteins: the GAL4 DNA-binding domain fused to CDK4,GAL4 db-CDK4; and the GAL4 activation domain fused to the proteinsencoded by HeLa cDNAs, GAL4ad-cDNA. YPB2 was used as the recipient yeastas it is a strain that contains two chromosomal genes under the controlof two different GAL4-dependent promoters: HIS3 and LacZ. YPB2 wastransformed with a mixture of two plasmids encoding, respectively, theGAL4 db-CDK4 and the GAL4ad-cDNA fusions; several clones were obtainedthat grew in the absence of histidine and that turned blue in thepresence of β-gal. From DNA sequencing data it was determined that eachof the positive clones derived from the same gene, although one grouprepresented mRNAs with a shorter 3′ end. The sequence of these cDNAscontained, in-phase with the GAL4ad, an open reading frame encoding aprotein of 148 amino acids with a predicted molecular weight of 15,845daltons (see SEQ ID NOs: 1 and 2). The sequence of p16 was compared bystandard methods with those present in the currently available databanks and no significant homologies were found.

To test if p16 would specifically bind CDK4, YPB2 were cotransformedwith the GAL4ad-p16 fusion as well as with several target GAL4 db fusionconstructs containing, respectively, cdc2, CDK2, CDK4, CDK5, PCNA andSnfl (a fission yeast kinase). Transformed cells were plated with andwithout histidine. Only the GAL4 db-CDK4 fusion interacted withGAL4ad-p16 to an extent which allowed growth in the absence ofhistidine, indicating that this pair of fusion proteins specificallyreconstituted a functional GAL4 activator able to enhance the expressionof the HIS3 gene. The same result was obtained when the ability totransactivate the expression of the β-galactosidase gene was assayed.

The specificity of this interaction was further demonstrated in acell-free system, by mixing in vitro translated ³⁵S-labeled CDKs with apurified bacterially-produced fusion protein consisting of glutathione-Stransferase (GST) linked to p16 (17). The GST-p16 fusion was recoveredby binding to glutathione-sepharose beads and the association of eachCDK was analyzed by gel electrophoresis. Consistent with the previousobservations, GST-p16 bound much more efficiently to CDK4 than to cdc2,CDK2 or CDK5.

Since the predicted molecular weight of p16 is close to 16 Kd, theidentity of p16 as the CDK4-associated p16 protein found in transformedcell lines (see above) was determined. Two in vitro translation productsof 15 KD and 17 KD were obtained from the p16 cDNA. These products, aswell as the CDK4-associated p16 protein from HeLa cells were treatedwith N-chlorosuccinimide. The partial NCS-proteolytic pattern of the 17KD cDNA-derived product was very similar to the pattern obtained withthe CDK4-associated p16 protein from HeLa cells, strongly suggestingthat the p16 cDNA actually corresponds to p16. Partial digestion with V8protease of the 17 KD cDNA-derived product and p16 also yielded similarpatterns. It is interesting to note that the p16 protein overexpressedin insect cells has an electrophoretic mobility of 15 KD, and its NCSproteolytic map is identical to that obtained with the 15 KD cDNAderived product. This suggests that the actual p16 found in human cellsand the 17 KD in vitro translation product correspond toposttranslationally modified proteins. The fact that the p16 proteinoverexpressed in insect cells interacts with CDK4 suggests that thismodification is not essential for the interaction (see below).

The identity between p16 and the CDK4-associated protein p16 was furtherconfirmed using antibodies raised against the purified GST-p16 fusionprotein. Several human cell lines were used for this experiment: anormal cell line WI38, derived from normal lung fibroblasts; the VA13cell line derived from WI38 by transformation with the SV40 T-antigen;and HeLa cells. As set out above, anti-CDK4 immunoprecipitates of WI38revealed the association of CDK4 with cyclin D1, PCNA, p21 and p16. Incontrast, in VA13 and HeLa cells CDK4 is only associated with p16.Anti-p16 immunoprecipitates contained a protein with an apparentmolecular weight of 16 KD which was readily detectable in the twotransformed cell lines, VA13 and HeLa but to a lesser extent in thenormal cell line WI38. This protein not only had the sameelectrophoretic mobility as the p16 protein coimmunoprecipitated withanti-CDK4 serum, but also had an identical NCS partial proteolyticpattern. In addition to p16 a protein of 33 Kd was observed in anti-p16coimmunoprecipitates that was shown to be identical to CDK4 byV8-proteolytic mapping.

Northern analysis of the transcripts present in WI38 and VA13 cellsindicated that the p16 mRNA was around many times less abundant in WI38cells compared to VA13 cells. This difference approximately correspondedto the observed difference in the amount of p16 protein between the twocell lines, suggesting the possibility that p16 expression might beregulated at a transcriptional or post-transcriptional level. Indeed, inthree non-virally transformed cell lines the expression of p16 could notbe detected even after overexposure of the gel.

To study the biochemical consequences of the interaction of p16 withCDK4, active CDK4-cyclin D complexes have been reconstituted in vitro bystandard protocols (Kato et al. Genes Der 7:331 (1993); and Ewen et al.Cell 73:487 (1993)). The three relevant components, CDK4, p16 and cyclinD1, were expressed in baculovirus-infected insect cells. Extracts wereprepared from metabolically ³⁵S-labeled insect cells that separatelyoverexpressed p16, CDK4 or cyclin D1, as well as from cellsoverexpressing both CDK4 and cyclin D1. In response to increasingamounts of p16, corresponding decreases in the ability of CDK4 tophosphorylate RB was observed. This inhibition correlated with theassociation between p16 and CDK4 as detected by immunoprecipitation. Noinhibition was observed when CDK2-cyclin D2 complexes were used in asimilar assay. To confirm that the inhibition of CDK4 was due to p16, aHis-tagged p16 fusion protein (His-p16) was created to have an aminoterminal extension of 20 amino acids containing a tract of 6 histidineresidues. This fusion protein was overexpressed in baculovirus-infectedinsect cells, and was purified by virtue of the high-affinityassociation of the histidine tract to nickel-agarose beads. The His-p16protein preparation was shown to be >90% pure, and inhibited theactivity of the CDK4-cyclin D1 complex under conditions similar to thoseused for inhibition by the whole lysates.

The role of the retinoblastoma gene product (RB), appears to be as acell-cycle checkpoint which appears to at least act be sequesteringtranscription factors responsible for the proteins of phase. In manycarcinomas, p53 function is lost by mutation or deletion. RB, on theother hand, is not apparently altered as often. However, because p16down-regulates the CDK4/cyclin D complex, which acts to phosphorylateRB, it is proposed herein that p16 loss in certain carcinomas canalleviate the effects of the RB checkpoint and, in some manner ofspeaking, represent a checkpoint deficiency analogous to p53 loss. Theloss of p16 would result in more effective phosphorylation of RB andhence would remove the RB-mediated inhibition of the cell-cycle.Consistent with this notion, it is described below that in a variety ofhuman tumor cells, such as cells which over-express a D-type cyclin,e.g. cyclin D1 or D2, the p16 gene is lost from the cell, e.g.homozygously deleted.

Moreover, as described in the examples below, the p16 gene was found tomap to the human region 9p21-22, a known melanoma locus (Wallcer et al.(1994) Oncogene 9:819; Coleman et al. (1994) Cancer Res 54:344; Cheng etal. (1993) Cancer Res 53:4761; and Cannon-Albright et al. (1992) Science258:1148). The chromosomal mapping was further confirmed by analysis ofsomatic cell hybrids through PCR amplification (using primers ex1A andex13 of FIG. 2A). Somatic hybrids containing human chromosome 9 resultedin positive PCR products being applified.

Utilizing primers generated from the cDNA sequence of human p16 (SEQ IDNO: 1) which are shown in FIG. 1, the genomic p16 gene was partiallysequenced to determine intron/exon boundaries. The approximate sequencesof the nucleic acid flanking Exon 1 and Exon 2 (see FIG. 1) are shown inFIGS. 2A and 2B and 3A-D respectively.

Genomic DNA was isolated from a variety of human tumor lines (Sambrooket al. Molecular Cloning: A Laboratory Manual, CSHL Press, Cold SpringHarbor, N.Y. (1989)) and was probed by PCR reactions for the presence orabsence of p16 sequences. In particular, primers ex1A and ex13 (FIG. 2A)were used to score for exon 1 of p16, and primers ex14 and ex15werelikewise used to detect exon 2 of p16. As shown in FIG. 4, the p16 geneis disrupted in several tumor cell lines, confirming that p16 is indeedlikely to be critical in cell transformation in certain cancerous cells.Moreover, probing of these cell lines with full length p16 cDNA (SEQ IDNO: 1) demonstrated that in at least 3 of those cells apparently missinga portion of the p16 gene, the entire gene was in fact absent.

Based on immunoprecipitation experiments with anti-p16 antibodies, aswell as oligonucleotide hybridization assays, it became apparent thatthe p16 protein represented by SEQ ID NO: 2 is merely one member of alarger family of related cell-cycle regulatory proteins. For instance,even under high stringency conditions, Southern hybridizationexperiments of mRNA from different tissue types has indicated thatapproximately 4 closely related transcripts are produced. These p16homologs, members of the CCR-protein family, may have arisen by geneduplication (e.g. each CCR-protein arises from a distinct gene) or fromalternate exon splicing at the mRNA level, or a combination thereof.

Utilizing a probe consisting of the coding region of the human p16 gene,we have screened a mouse embryonal stem cell library and have isolated agenomic clone containing the coding region for a mouse homolog of thehuman p16-genie described above. This clone was isolated under low tomoderate stringency conditions (1×SSC at 50° C.). This DNA (14 kB) hasbeen cloned in two independent pieces and the restriction map for ninerestriction endonucleases has been performed (see FIG. 5). The mouseCCR-gene has been completely sequenced and the coding region isapparently made up of only two exons that have been located in therestriction map by Southern hybridization. The apparent molecular weightof the mouse CCR-protein is 13.5 kDa, and the nucleic acid and aminoacid sequence of the mouse CCR-protein, termed herein “p13.5”, is givenin SEQ ID NO: 5 and 6 respectively.

Moreover, utilizing degenerate probes based on the most highly conservedsequences between the human p16 clone and mouse p13.5 clone (e.g.between residues Met-52 through Gly-135 of human p16 and Met-1 throughGly-83 of mouse p13.5), we have isolated a number of human p16 homologs.

In addition, it has been noted that TGF-β treatment causes accumulationof RB in the under-phosphorylated state and expression ofRB-inactivating viral oncoproteins prevents TGF-β induced cell cyclearrest (Laiho et. al. (1990) Cell 62:175-185; and Pietenpol et al.(1990) Cell 61:777-785). While prior publications have suggested thatTGF-β treatment results in down-regulation of CDK4 expression (Ewen etal. (1993) Cell 74:1009-1020), the data suggested to us that TGF-β mightfunction through suppression of RB phosphorylation and pointed to thepossibility that one result of TGF-β treatment might be inhibition ofcyclin dependent kinases.

Accordingly, to investigate the mechanism by which TGF-β inhibits cellproliferation, we examined anti-CDK immunoprecipitates from humankeratinocytes which had been arrested in G₁ by exposure to TGF-β.Notably, immunoprecipitates of two G₁-specific cyclin kinases, CDK4 andCDK6, contained several low molecular weight, associated proteins. Theseincluded p16 and two additional proteins of approx. 15 and 15.5 kDa.These proteins were not recovered in parallel CDC2 or CDK2immunoprecipitates but were recovered in anti-p16 immunoprecipitates,suggesting that p15, p15.5 and p16 might be related. This was confirmedby western blotting of CDK4 and CDK6 immunoprecipitates whichdemonstrated that p15 and p15.5 were weakly cross-reactive with the p16antiserum.

To isolate clones encoding putative p16 relatives, we constructed a cDNAlibrary from TGF-β arrested HaCaT cells (Boukamp et al. (1988) J CellBiol 106:761-771) and probed this library at low-stringency with the p16coding sequence (SEQ ID NO: 1). One clone obtained in this screenencoded a 137 amino acid protein (predicted M.W. 14.7 kDa.) withhomology to p16. Based upon this homology and upon biochemicalproperties described below, we have named this protein p15. The first 50amino acids of p15 and p16 share approx. 44% identity. This is followedby an 81 amino acid region of approx. 97% identity (see FIG. 6) afterwhich p15 and p16 sequences diverge. The sequence of p15 can be dividedinto four ankyrin repeats suggesting that this structural motif isconserved in the CCR-protein family. In vitro translation of the p15cDNA produced a protein which precisely comigrated with the p15 bandpresent in CDK4, CDK6 and p16 immunoprecipitates from TGF-β arrestedHaCaT cells. Identity of these proteins was confirmed by protease andchemical cleavage mapping.

To investigate the functional similarity of p15 and p16, we expressedp15 as a fusion protein in bacteria and tested its ability to bind andinhibit cyclin dependent kinases. p15 specifically bound CDK4 and CDK6but did not appreciably bind CDC2, CDK2 or CDK5. To assess theconsequences of binding, p15 was added to active cyclin/CDK complexesexpressed in insect cells. p15 specifically inhibited the cyclin D/CDK4and cyclin D/CDK6 enzymes but had no effect on CDK2/cyclin A kinase.Thus p15 is a functional member of the CCR-protein family. Moreover,FISH mapping of the p15 gene demonstrated that this gene lies adjacentto the p16 gene at 9p21.

While we first noted p15 in immunoprecipitates from HaCaT cells whichhad been arrested in G₁ by serum starvation and re-stimulation in thepresence of TGF-β, by comparison, we found that asynchronous, rapidlyproliferating HaCaT cells contained considerably lower levels of p15 inCDK4 and CDK6 immunoprecipitates. To separate effects of TGF-β.treatment from effects of G₁ arrest, asynchronous cultures were treatedwith TGF-β. for various periods, after which patterns of CDK4 and CDK6associated proteins were examined. In as little as four hours followingTGF-β addition, p15 levels rose in CDK4 and CDK6 immunoprecipitates,reaching peak levels after 6-8 hours. In contrast, CDK-associated p16levels were unaffected by TGF-β. Northern blotting of RNA from culturestreated in parallel revealed that increased CDK4-associated p15reflected increased abundance of p15 mRNA. In 2 hours following TGF-βtreatment, p15 mRNA began to rise and reached a peak induction ofapprox. 30-fold after 6-8 hours. In contrast, p16 mRNA levels did notvary.

Two other mechanisms for TGF-β mediated cell cycle arrest have beenpreviously proposed. In Mv1Lu cells, TGF-β treatment suppressed CDK4synthesis. This was deemed causal since cells could be renderedresistant to TGF-β by constitutive overexpression of CDK4. In HaCaTcells, TGF-β treatment had no effect on CDK4 protein or mRNA levels.Based upon the properties of p15, we would predict that CDK4overexpression could also render HaCaT cells TGF-β resistant bytitrating the p15 CDK4/CDK6 inhibitor. p27, a CDK inhibitor which waspurified from TGF-β arrested cells, has also been proposed as a linkbetween TGF-β and cell cycle control. However, in HaCaT cells, TGF-βtreatment had no effect on p27 mRNA levels. Thus any contribution thatp27 may make to TGF-β mediated cell cycle arrest in these cells mustoccur by regulation at the post-translational level.

Considered together, our data suggest that p15 may function as aneffector of TGF-β mediated cell cycle arrest via inhibition of CDK4 andCDK6 kinases. p15 may be the sole mediator of TGF-β induced arrest insome cells, or may cooperate with other TGF-β responsive pathways. TGF-βcan regulate differentiation in some cell types, and the ability ofTGF-β to affect cell cycle progression through p15 may also contributeto these processes.

Moreover, cytogenetic abnormalities at 9p21 are common in many types ofhuman tumors suggesting the presence of a tumor suppressor gene at thislocus. An inherited cancer syndrome which causes predisposition tomelanoma also maps to 9p21. In addition to our data presented herein,and in U.S. Ser. No. 08/248,812 and in 08/227,371, p16 was initiallyproposed as a candidate for both of these activities based upon analysisof p16 deletions and point mutations in cell lines. However, thepresence of a second functional member of the p16 family at 9p21 raisesthe possibility that loss of tumor suppression may involve inactivationof either or both genes. The response of p16 to viral oncoproteinsindicates that it may function in intracellular growth regulatorypathways, while results presented here suggest that p15 may transduceextracellular growth inhibitory signals. Thus deletions of 9p21 whichremove both genes (or other mutations that might inactivate both) couldsimultaneously negate two major proliferative control pathways. In thisregard, the ability of TGF-β to induce growth arrest is reduced or lostin many neoplastically transformed cell lines. In particular,melanocytes are sensitive to growth inhibition by TGF-β, but manymetastatic melanoma cells are TGF-β resistant.

Example 1 Demonstration of Selective Subunit Rearrangement of Cell-CycleComplexes in Association with Cellular Transformation by a DNA TumorVirus or its Oncogenic Product

(i) Cellular Transformation with DNA Tumor Virus SV40 is Associated withSubunits Rearrangement of Cell-Cycle Complexes

Preparation of [³⁵S] methionine-labelled cell lysates and polyacrylamidegel electrophoresis were as described above, as well as described in PCTPublication No. WO92/20796. Cell lysates were prepared from either humannormal diploid fibroblast cells WI38 or DNA tumor virus SV40 transformedWI38 cells, VA 13. Cell lysates were immunoprecipitated with antibodiesagainst each cell-cycle gene products.

(ii) Subunit Rearrangements of Cell-Cycle Complexes in Two DifferentPair Cell Lines

Methods for preparation of cell lysates are the same as described above.Two different pair cell lines were used in these experiments. HSF43 is anormal human diploid fibroblast cell line and CT10 (full nameCT10-2C-T1) is a derivative of HSF43 transformed by SV40 large tumorantigen. CV-1 is an African green monkey kidney cell line and COS-1 is aderivative of CV-1 transformed by SV40.

(iii) Cellular Transformation by DNA Tumor Virus SV40 is Associated withRearrangement of PCNA Subunit of Cell-Cycle Complexes

Preparation of Cell Lysate, Electrophoresis, and Western BlottingConditions are the same as described above. Normal human diploidfibroblast cell lines and their SV40 transformed cell lines aredescribed above. Immunoprecipitates derived from each antibody wereseparated on polyacrylamide gels and blotted with anti-PCNA antibody.

(iv) Cellular Transformation by DNA Tumor Virus Sv40 is Associated withRearrangement of CDK4 Subunit of Cell-Cycle Complexes

Preparation of Cell Lysate, Electrophoresis, and Western BlottingConditions are the same as previously described. Normal human diploidfibroblast cell lines and their SV40 transformed cell lines aredescribed above. Immunoprecipitates derived from each antibody wereseparated on polyacrylamide gels and blotted with anti-CDK4 antibody.

Example 2 Cloning of p16, an Inhibitor of CDK4 Activity

(i) Cloning of p16 Using the Two Hybrid Assay

Saccharomyces cerevisiae YPB2 cells were transformed simultaneously witha plasmid containing a GAL4 db-p16 fusion and with a plasmid containing,respectively, the GAL4ad fused to cdc2 (CDK1), CDK2, CDK4, CDK5, PCNA(proliferating cell nuclear antigen), and the fission yeast kinaseSnf 1. After growing cells in medium selective for both plasmids (minustryptophan and minus leucine), two colonies were picked randomly andwere streaked in plates that either contained or lacked histidine. Theability to grow in the absence of histidine depends on the expression ofthe HIS3 gene that is under a GAL4-responsive promoter and, therefore,indicates that a functional GAL4 activator has been reconstitutedthrough the interaction of p16 with the corresponding target protein.

(ii) Interaction of p16 CDKs

Purified bacterially-produced GSTp16 fusion protein was mixed with³⁵S-labeled in vitro translated cdc2, CDK2, CDK4 and CDK5. Mixturescontained 0.5 μg of purified GST-p16 and an equivalent amount of invitro translated protein (between 0.5 to 5 μl; TNT Promega) in a finalvolume of 200 μl of a buffer containing 50 mM Tris-HCl pH 8, 120 mM NaCland 0.5% Nonidet P-40. After 1 h at 4° C., 15 μl of glutathione-agarosebeads were added and incubation was resumed for an additional hour.Beads were recovered by centrifugation, washed 4 times with theincubation buffer, and mixed with standard protein-gel loading buffer.Samples were loaded into a 15% poly-acrylamide gel and ³⁵S-labeledproteins were detected by fluorography. The GSTp16 fusion protein wasoverexpressed in the pGEX-KG vector and purified by standard techniques.The in vitro translation templates were derived from the pBLUESCRIPT®vector (Stratagene).

(iii) Proteolytic Mapping of p16

The in vitro translated S³⁵-labeled p16 (TNT Promega) was obtained usingthe p16 cDNA cloned into pBLUESCRIPT® vector (Stratagene) as a template,and the CDK4-associated p16 protein was co-immunoprecipitated with ananti-CDK4 serum from metabolically S³⁵-labeled HeLa cells lysates.Partial proteolysis was done over the corresponding gel slices afterextensive equilibration in a buffer and digestion was accomplished byaddition of NCS at different concentrations. The products were run in a17.5% polyacrylamide gel and detected in a phosphorimager FUJIX® 2000.

(iv) Detecting the Effects of p16 on CDK4-Cyclin D Complexes

Baculovirus-infected insect cells overexpressing p16, CDK4, cyclin D1,or both CDK4 and cyclin D1 together were metabolically ³⁵S-labeled. Thedifferent incubation mixtures were composed by extracts containing p16,CDK4, cyclin D1 and both CDK4 and cyclin D1, and were immunoprecipitatedwith anti-p16 serum, anti-CDK4 serum without any previous preincubation,and anti-CDK serum preincubated with the peptide originally used toraise the antiserum and anti-cyclin D1 serum. Immunoprecipitates werethen analyzed by SDS-PAGE.

Example 3 Chromosomal Mapping of p16

Genomic clones of the human p16 gene were isolated by stringencyscreening (68° C. with 0.1×SSC wash) of a λFIXII human genomic library(Strategene) with cDNA probes. Isolated phage clones were confirmed byhigh stringency Southern hybridization and/or partial sequence analysis.Purified whole phage DNA was labelled for fluorescent in situhybridization (FISH) analysis.

FISH analysis was performed using established methods (Demetrick et al.(1994) Cytogenet Cell Genet 66:72-74; Demetrick et al. (1993) Genomics18:144-147; and DeMarini et al. (1991) Environ Mol Mutagen 18:222-223)on methotrexate or thymidine synchronized, phytohaemagglutininstimulated, normal peripheral blood lymphocytes. Suppression with amixture of sonicated human DNA and cotl DNA was required to reduce thebackground. The stained slides were counterstained with propidium iodide(for an R banding pattern) or DAPI and actinomycin D (for a DA-DAPIbanding pattern), mounted in antifade medium and visualized utilizing aZeiss AXIOPHOT™ microscope. Between 30 and 100 mitoses were examined foreach gene location. Photographs were taken using a cooled CCD camera.Alignment of three color fluorescence was done under directvisualization through a triple bandpass filter (FITC/Texas Red/DAPI).The p16 gene was visualized to map to 9p21-22.

Example 4 Cloning of Mouse CCR-Proteins

Utilizing a probe consisting of the coding region of the human p16 gene,we have screened a mouse embryonal stem cell library and have isolated agenomic clone containing the coding region for a mouse homolog of thehuman p16 gene described above. This clone was isolated under low tomoderate stringency conditions (1×SSC at 50° C.). This DNA (14 kB) hasbeen cloned in two independent pieces and the restriction map for ninerestriction endonucleases has been performed (see FIG. 5). The mouseCCR-gene has been completely sequenced and the coding region isapparently made up of only two exons that have been located in therestriction map by Southern hybridization. The apparent molecular weightof the nouse CCR-protein is 13.5 kDa, and the nucleic acid and aminoacid sequence of the mouse p16 homolog, termed herein “p13.5”, is givenin SEQ ID NO: 5 and 6 respectively.

Utilizing a probe based on the nucleotide sequence of the conservedregion of Met1-Arg80 (see FIG. 6) of the mouse p13.5 clone, a p19embryonal carcinoma library (Stratagene) was probed under highstringency conditions. Several clones were isolated from the library,some of which were seemingly identical to the p13.5 clone, and one whichthe partial sequence (SEQ ID NOs: 7 and 8) indicated that it was themurine homolog of either p16 or p15.

Example 5 Cloning of p15 from Human Cells

The sequence of the p15 cDNA is shown along with the deduced amino acidsequence of the protein in SEQ ID NOs: 3 and 4 respectively. The deducedamino acid sequence of p15 was compared to that of p16 (e.g. see FIG.6), and areas of homology were identified using the BLAST program.

(i) Cell Culture

HaCaT cells were routinely maintained in DMEM containing 10% fetalbovine serum (FBS) (Boukamp et al. (1988) J Cell Biol. 106:761-771). ForTGF-β arrest, HaCaT cells were grown to confluence in DMEM containing10% FBS (FBS) and then serum starved for 3 days in DMEM containing 0.1%FBS. Cells were re-stimulated by addition of new media containing 10%FBS and 2 ng/ml TGF-β (purified from human platelets, Calbiochem). Forlibrary construction, RNA was prepared 22 hours after re-stimulation.For immunoprecipitations experiments, cells were labelled with³⁵S-methionine in the presence of TGF-β for four hours beginning at 19hours after re-stimulation.

(ii) Library Construction and Screening

RNA was prepared from TGF-β treated cells and from cells that were serumstarved and then re-stimulated in the absence of TGF-β using RNAZOL Baccording to the manufacturer's instructions. The cDNA library which wasused to isolate the p15 clone was constructed from a mixture of RNAderived from treated and untreated cells. Messenger RNA preparation andcDNA synthesis were exactly as previously described (Hannon et al.(1993) Genes Dev. 7:2378-2391). Double-stranded cDNA was ligated intoλ-ZapII arms according to the manufacturers instructions. Low stringencyhybridization was performed at 50° C. in 500 mM NaPO₄, pH 7.0, 1 mMEDTA, 15% Formamide, 7% SDS, 0.1% bovine serum albumin (wash: 1×SSC, 50°C.). The p15 cDNA was also isolated from a human mammary epithelial celllibrary. Cell lysis, immunoprecipitation and protease mapping wereperformed exactly as previously described (Xiong et al. (1993) Nature366:701-704). For chymotrypsin digestion, either 7 μg or 1.5 μg ofenzyme was used. Gels for chymotrypsin mapping contained 0.1% SDS.

Example 6 p15 is a Specific Inhibitor of CDK4 and CDK6

Preparation of GST-p16 fusion protein from bacteria is described above.GST-p15 was prepared identically.

(i) p15 binds CDK4 and CDK6

In vitro translated CDKs were prepared using the TNT-lysate in vitrotranslation kit (Promega) according to the manufacturer's instructions.For the binding assay, GST-p15 or GST-p16 (250 ng) was incubated with invitro translated CDKs, e.g. ³⁵S-labelled CDC2, CDK2, CDK4, CDK5 or CDK6,for 30 minutes at 30° C. in 30 μl containing 20 mM Tris, pH 8.0, 10 mMMgCl₂, 1 mM EGTA. Following incubation, mixtures were diluted to 250 μlin IP buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% NP-40) and incubatedfor 1 hour with 12.5 μl of glutathione sepharose. Bound proteins wererecovered on glutathione sepharose, washed four times with 1 ml of IPbuffer, and then released by boiling in SDS gel sample buffer. Boundproteins were analyzed by electrophoresis in a 17.5% PAGE gel. Forcomparison, a similar experiment using GST-p16 was carried out.

(ii) p15 Binding Inhibits Cyclin D/CDK4 Kinase

Active cyclin D/CDK4 kinase, present in lysates of baculovirus infectedinsect cells, was incubated with increasing quantities of GST-p15 for 30minutes at 30° C. Preparation of baculovirus lysates containing activecyclin D/CDK4 kinase is described above. Lysates containing activecyclin D/CDK6 were prepared identically. For kinase assays 10 μl ofbaculoviral lysates were mixed with approximately 0, 10 ng, 20 ng, 50ng, or 100 ng of GST-p15 or GST-p16 and incubated for 30 minutes at 30°C. in a total volume of 30 μl containing 20 mM Tris, pH 8.0, 10 mMMgCl₂, 1 mM EGTA. Following incubation, 0.5 μg GST-RB and 0.5 μl ofλ³²P-ATP (5 μCi, 3000 Ci/mMol) were added for 10 minutes at 30° C. assubstrates. Reactions were stopped by the addition of 250 μl of IPbuffer and 15 μl of glutathione sepharose. After a further 1 hour ofincubation at 4° C., beads were washed four times with IP buffer beforerelease of bound proteins and subsequent electrophoresis. Like p16, thep15-GST fusion protein was able to inhibit the kinase activity of CDK4.

(iii) p15 Inhibits Cyclin D/CDK6 Kinase

Cyclin D/CDK6 complexes were produced in baculovirus infected insectcells and incubated with increasing quantities of GST-p15 or GST-p16 asdescribed above. Following incubation, the ability of these complexes tocatalyze GST-RB phosphorylation was determined. Both p15 and p16demonstrated an ability to inhibit the CDK6 kinase activity.

Example 7 TGF-β Treatment Increases Association of p15 with CDK4 andCDK6

HaCaT cells were cultured as described above. TGF-β treatment wasinitiated by adding TGF-β, to existing media to 2 ng/ml. For the lasttwo hours of TGF-β treatment, cells were labelled with ³⁵S-methionine.For cell labelling, media was changed to DMEM minus methionine(Gibco-BRL) containing 2 ng/ml of TGF-β, 10% dialyzed FBS and 0.5 mCi/mlof ³⁵S-methionine/cysteine (trans-label, NEN). Cells were lysed andproteins were immunoprecipitated using either anti-CDK4 or anti-CDK6antibodies. Cell lysis and immunoprecipitation were as described above.HaCaT cultures treated with TGF-β in parallel were stained with DAPI andanalyzed by FACS. For 8 hours following TGF-β addition, there was noappreciable change in the percentage of G₁ cells. After fourteen hours,the G₁ population began to increase. Arrest of HaCaT cells in G₁(approx. 85% G₁ cells) required at least 24 hours after treatment of anasynchronous culture.

Example 8 TGF-β Treatment Induces p15 mRNA

Asynchronous HaCaT cultures were treated with TGF-β for the indicatedtimes. Total RNA was prepared from treated cells and used for Northernblotting with a probe specific for p15 (e.g. probes consisted of thefirst coding exons of this genes and was prepared by PCR). Hybridizationused for northern blots consisted of 200 mM NaPO₄, pH 7.0, 1 mM EDTA,15% Formamide, 7% SDS, 0.1% bovine serum albumin at 65° C.; wash:0.2×SSC, 65° C. The p15 probe recognized three p15 mRNAs ofapproximately 0.8, 2.2 and 3.2 Kb. Northern signals were quantitated ona Fuji BAS2000 phosphorimager and plotted to give a graphicalrepresentation of the results. RNA amounts were normalized by mass, andover-probing of the blot with a human actin probe suggested no more than10% variance in RNA amounts between lanes.

RNAs identical to those used in p15 probe panel were also probed with afragment specific for p16. Furthermore, RNA from TGF-β treated HaCaTcells was probed oligonucleotides derived from either a fragment ofhuman CDK4 coding sequence or a fragment of the p27 cDNA.

Example 9 Generating a Transgenic Mouse p13.5 Knockout

The disruptive construct is formed by the two DNA regions of approx. 3-4Kb flanking the p13.5 gene. These DNA pieces are cloned at both sides ofa gene marker that will be used to select the mouse embryonal stem (ES)cells that have incorporated this DNA after transfection. Regions whichare homologous to the p16 locus are in turn flanked by another markerwhich allows selection against cells which have incorporated thedisruption vector by non-homologous recombination (e.g. at a locus otherthan that of the mouse p13.5 gene). Those cells where insertion hasoccurred in the appropriate position are injected into mouse blastocytesand implanted into the appropriate female mice following standardprotocols (Manipulating the Mouse Embryo, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1986; and Jaenisch (1988) Science240:1468-1474.)

Chimeric pups resulting from the engineered blastocysts can beidentified by a coat color marker specific to the transfected ES cells(agouti). Mice with high degrees of chimerism are crossed to identifythose with chimeric germ lines and to generate non-chimeric heterozygousdisruptants. Homozygous disruptants are derived by breeding thenon-chimeric heterozygotes.

Example 10 Characterization of CCR-Insensitive CDK4 Mutants

A mutant of CDK4 was discovered in cells from a melanoma patient, andthe sequence of the mutant enzyme was determined. From the sequence, itwas determined that that the gene contained a Arg.fwdarw.Cys at position24 (see SEQ ID NO: 9).

We reisolated the mutant by cloning CDK4 (see Matsushime et al. (1992)Cell 71:323-334) and generated a the Cysteine mutation byoligonucleotide primer mutagenesis. To characterize the effect of themutation, we compared the mutant and wild-type enzyme based on a numberof different criteria, including intrinsic activity (e.g. did the mutantconstitutively activate CDK4), as well as the ability of otherregulatory proteins to control CDK4 activation. Briefly, we generated aseries of baculovirus expression systems for over-expressing variousproteins. In particular, Sf9 cell lysates (Desai et al. (1992) Mol CellBiol 3:571-582, and Example 6 above) were obtained for mutant andwild-type CDK4, cyclin D1, p16, p15, p21 and p27 (see Polyak et al.(1994) Genes Dev 8:9-22; and Toyoshima et al. (1994) Cell 78:67-74).Using a GST-RB fusion protein (Example 6) as a substrate for detectingCDK4 kinase activity, various combinations of lysate were lixed andtested for CDK4 activation/inhibition.

When the mutant CDK4 was expressed alone in Sf9 cells, no appreciablephosphorylation of the RB substrate was detected, as is also the casewith the wild-type enzyme, indicating that the mutation did causeconstitutive activation of CDK4. Overexpression of a CDK4 and cyclin D1in an Sf9 lysate was also identical for both mutant and wild-typekinase, as each was shown to be activated in the presence of cyclin D1.However, upon addition of increasing amounts of either p16- or p15containing lysate to the CDK4/cyclin D mixture, the wild-type CDK4 wasinhibited yet the mutant CDK4 was relatively unaffected, indicating thatthe mutation gave rise to kinase whose activity is insensitive to eitherp15 or p16. Furthermore, immunoprecipitation demonstrated that neitherp15 or p16 were capable of binding the mutant, as they were apparentlylost from the complex which is ordinarily seen with the wild-type CDK4.Finally, similar experiments carried out with p21 and p27 indicated thatthe particular mutation, Arg24-Cys, did not effect the binding orinhibitory ability of either of those proteins. An analogous mutation toArg31 of CDK6 (SEQ ID NO: 10; and Bates et al. (1994) Oncogene 9:71-79for the wild-type gene) is expected to have the same effect.

All of the above-cited references and publications are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A kit for detecting the level of cyclin-dependent kinase inhibitorp16 gene expression comprising an antibody specifically immunoreactivewith a 16 kD protein that co-precipitates with CDK4 from cell lysates ofSV40-transformed WI38 cells in the presence of an anti-CDK4 antibody. 2.The kit according to claim 1, wherein the antibody is a monoclonalantibody.
 3. The kit according to claim 1, wherein the antibody isimmunoreactive with a p16 protein comprising SEQ ID NO:
 35. 4. The kitaccording to claim 1, which is used for early diagnosis of neoplastic orhyperplastic disorders.
 5. The kit according to claim 1, which is a kitfor immunohistochemical staining.